Rupestris stem pitting associated virus nucleic acids, proteins, and their uses

ABSTRACT

The present invention relates to an isolated protein or polypeptide corresponding to a protein or polypeptide of a  Rupestris  stem pitting associated virus. The encoding DNA molecule, either alone in isolated form, in an expression system, a host cell, or a transgenic grape plant, is also disclosed. Other aspects of the present invention relate to a method of imparting  Rupestris  stem pitting associated virus resistance to grape plants by transforming them with the DNA molecule of the present invention, and a method of detecting the presence of a  Rupestris  stem pitting associated virus, such as RSPaV-1, in a sample.

This application claims the benefit of U.S. Provisional Patent Applications Ser. No. 60/047,147, filed May 20, 1997, and 60/069,902, filed Dec. 17, 1997.

This work was supported by the U.S. Department of Agriculture Clonal Repository—Geneva, Grant Nos. 58-2349-9-01 and 58-2349-9 and U.S. Department of Agriculture Cooperative Agreement Grant Nos. 58-1908-4-023, 58-3615-5-036, and 58-3615-7-060. The U.S. Government may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to Rupestris stem pitting associated virus (“RSPaV”) proteins, DNA molecules encoding these proteins, and diagnostic and other uses thereof.

BACKGROUND OF THE INVENTION

The world's most widely grown fruit crop, the grape (Vitis sp.), is cultivated on all continents except Antarctica. However, major grape production centers are in European countries (including Italy, Spain, and France), which constitute about 70% of the world grape production (Mullins et al., Biology of the Grapevine, Cambridge, U.K.:University Press (1992)). The United States, with 300,000 hectares of grapevines, is the eighth largest grape grower in the world. Although grapes have many uses, a major portion of grape production (˜80%) is used for wine production. Unlike cereal crops, most of the world's vineyards are planted with traditional grapevine cultivars, which have been perpetuated for centuries by vegetative propagation. Several important grapevine virus and virus-like diseases, such as grapevine leafroll, corky bark, and Rupestris stem pitting (“RSP”), are transmitted and spread through the use of infected vegetatively propagated materials. Thus, propagation of certified, virus-free materials is one of the most important disease control measures. Traditional breeding for disease resistance is difficult due to the highly heterozygous nature and outcrossing behavior of grapevines, and due to polygenic patterns of inheritance. Moreover, introduction of a new cultivar may be prohibited by custom or law. Recent biotechnology developments have made possible the introduction of special traits, such as disease resistance, into an established cultivar without altering its horticultural characteristics.

Many plant pathogens, such as fungi, bacteria, phytoplasmas, viruses, and nematodes can infect grapes, and the resultant diseases can cause substantial losses in production (Pearson et al., Compendium of Grape Diseases, American Phytopathological Society Press (1988)). Among these, viral diseases constitute a major hindrance to profitable growing of grapevines. About 34 viruses have been isolated and characterized from grapevines. The major virus diseases are grouped into: (1) the grapevine degeneration caused by the fanleaf nepovirus, other European nepoviruses, and American nepoviruses, (2) the leafroll complex, and (3) the rugose wood complex (Martelli, ed., Graft Transmissible Diseases of Grapevines, Handbook for Detection and Diagnosis, FAO, UN, Rome, Italy (1993)).

Rugose wood (RW) complex is a term to describe a group of graft-transmissible diseases which are important and widespread on grapevines grown world-wide. Symptoms of RW are characterized by pitting, grooving, or distortion to the woody cylinder of the grapevine scion, rootstock, or both. Based on symptoms developed on different indicator plants after graft inoculation, RW complex can be divided into four components: Kober 5BB stem grooving (KSG), LN 33 stem grooving (LNSG), grapevine corky bark (GCB), and Rupestris stem pitting (RSP) (Martelli, “Rugose Wood Complex,” in Graft-Transmissible Diseases of Grapevines. Handbook for Detection and Diagnosis, pp.45-54, Martelli, ed., Food and Agriculture Organization of the United Nations, Rome, Italy (1993)). Because RW can cause severe decline and death to grapevines (Savino et al., “Rugose Wood Complex of Grapevine: Can Grafting to Vitis Indicators Discriminate Between Diseases?”, in Proceedings of the 9^(th) Meetings of the International Council for the Study of Viruses and Virus Diseases of the Grapevine, Anavim, Israel (1989); Credi and Babini, “Effect of Virus and Virus-like Infections on the Growth of Grapevine Rootstocks,” Adv. Hort. Sci., 10:95-98 (1996)), it has been included in healthy grapevine detection schemes used in major grapevine growing countries including Italy, France, and the United States.

RSP was discovered in California in the late 1970s (Prudencio, “M. Sc. Thesis: Comparative Effects of Corky Bark and Rupestris Stem Pitting Diseases on Selected Germplasm Lines of Grapes,” University of California, Davis, California, 36 pages (1985); Goheen, “Rupestris Stem Pitting,” in Compendium of Grape Diseases, p. 53, Pearson and Goheen, eds., American Phytopathological Society Press, St. Paul, Minn., USA (1988) (“Goheen”)). The disease was defined by Goheen as follows: after graft inoculation with a chip bud from an infected grapevine, the woody cylinder of the indicator plant Vitis rupestris Scheele St. George (“St. George”) develops a narrow strip of small pits extending from the inoculum bud to the root zone. Grafted St. George plants were checked for wood symptoms 2 to 3 years after inoculation. In contrast to GCB, which elicits pitting and grooving on St. George and LN 33, RSP does not produce symptoms on the latter (Goheen, “Rupestris Stem Pitting,” in Compendium of Grape Diseases, p. 53, Pearson and Goheen, eds., American Phytopathological Society Press, St. Paul, Minn., USA (1988)).

RSP is probably the most common component of the RW complex on grapevines. Surveys in California revealed a high disease incidence in many grapevine cultivars imported from Western Europe and Australia (Goheen, “Rupestris Stem Pitting,” in Compendium of Grape Diseases, p. 53, Pearson and Goheen, eds., American Phytopathological Society Press, St. Paul, Minn., USA (1988)). An examination of indexing records in California compiled over 23 years revealed RSP infection in 30.5% of 6482 grapevine selections introduced from around the world (Golino and Butler, “A Preliminary Analysis of Grapevine Indexing Records at Davis, California,” in Proceedings of the 10th Meeting of the ICVG, pp. 369-72, Rumbos et al., eds., Volos, Greece (1990)). Indexing in New York State showed that 66% of 257 grapevines tested on St. George developed typical small pits below the inoculum bud or around the woody cylinder (Azzam and Gonsalves, Abstract: “Survey of Grapevine Stem-Pitting in New York and Isolation of dsRNA from a Grapevine Selection Infected with Stem Pitting,” Phytopathology 78:1568 (1988)). Furthermore, several reports have indicated that RSP is the most frequently detected component of the RW complex in Italy (Borgo and Bonotto, “Rugose Wood Complex of Grapevine in Northeastern Italy: Occurrence of Rupestris Stem Pitting and Kober Stem Grooving,” in Extended Abstracts of the 11 th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine (ICVG), pp. 61-62, Gugerli, ed., Montreux, Switzerland (1993); Credi, “Differential Indexing Trials on Grapevine Rugose Wood Syndrome,” Extended Abstracts of the 11th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine (ICVG), p. 63, Gugerh, P., ed., Montreux, Switzerland (1993)).

The effect of RSP on growth, yield, and grapevine quality is not well understood and, thus, subject to debate. The reason for this ambiguity is the absence of a rapid and sensitive diagnostic tool. RSP is the most difficult grapevine disease to diagnose. Serological or molecular methods are not available for diagnosing RSP. Biological indexing on St. George, as described above, has remained the only approach to diagnose RSP. Biological indexing is labor intensive, time consuming (i.e., often requiring up to about three years to obtain results), and, by its very nature, subjective. Moreover, symptoms on St. George can be variable and not exactly as those defined by Goheen. In particular, Credi, “Characterization of Grapevine Rugose Wood Sources from Italy,” Plant Disease, 82:1288-92 (1997), recently showed that some RSP infected grapevines induced pitting that is restricted to below the inoculum bud, while others induced pitting around the woody cylinder of inoculated St. George. Thus, the present method of identifying the presence of RSP is not entirely adequate.

The etiology of RSP is unknown. Efforts to isolate virus particles from RSP-infected grapevines and to mechanically transfer the causal virus(es) to herbaceous host plants failed (Azzam and Gonsalves, “Detection of in Grapevines Showing Symptoms of Rupestris Stem Pitting Disease and the Variabilities Encountered,” Plant Disease, 75:96-964 (1991)). However, a major dsRNA species of ca. 8.3 kb, accompanied by a smaller dsRNA of ca. 7.6 kb, was consistently isolated from one Pinot Gris and four Pinot Noir clones that had been indexed positive for RSP (Walter and Cameron, “Double-Stranded RNA Isolated from Grapevines Affected by Rupestris Stem Pitting Disease,” Am. J. of Enology and Viticulture, 42:175-79 (1991)). In addition, a third dsRNA of ca. 5.5 kb was observed in three clones. Likewise, an apparently similar dsRNA species of ca. 8.0 and 6.7 kbp was isolated from dormant canes of RSP-infected grapevines collected from California, Canada, and New York (Azzam and Gonsalves, “Detection of dsRNA in Grapevines Showing Symptoms of Rupestris Stem Pitting Disease and the Variabilities Encountered,” Plant Disease, 75:960-64 (1991)). Six of eight Californian and three of five Canadian samples contained these two dsRNA species. However, results of New York samples were not consistent. Among eight RSP infected grapevine selections tested, only one showed these two dsRNAs. Using explants growing in tissue culture as source materials, dsRNA of ca. 359 bp was isolated from 21 of 31 grapevine cultivars, all of which were previously indexed on St. George and considered to be infected with RSP (Monette et al., “Double-Stranded RNA from Rupestris Stem Pitting-Affected Grapevines,” Vitis 28:137-44 (1989)).

In view of the serious risk RSP poses to vineyards and the absence of an effective treatment of it, the need to prevent this affliction continues to exist. Moreover, the absence of a rapid and accurate diagnostic assay prevents proper identification of RSP. The present invention is directed to overcoming these deficiencies in the art.

SUMMARY OF THE INVENTION

The present invention relates to an isolated protein or polypeptide corresponding to a protein or polypeptide of a RSP virus. The encoding RNA molecule or DNA molecule, in either isolated form or incorporated in an expression system, a host cell, or a transgenic Vitis scion or rootstock cultivar, are also disclosed.

Another aspect of the present invention relates to a method of imparting RSP virus resistance to Vitis scion or rootstock cultivars by transforming them with a DNA molecule encoding the protein or polypeptide corresponding to a protein or polypeptide of a RSP virus.

The present invention also relates to an antibody or binding portion thereof or probe which recognizes proteins or polypeptides of the present invention.

Still another aspect of the present invention relates to diagnostic tests which involve methods for detecting the presence of a RSP virus in a sample. The methods include the use of an antibody or binding portion of the present invention (i.e., in an immunoassay), or a nucleic acid probe obtained from a DNA molecule of the present invention (i.e., in a nucleic acid hybridization assay or gene amplification detection procedure). The antibody or binding portion thereof, or nucleic acid probe, is introduced into contact with the sample, whereby the presence of Rupestris stem pitting virus in the sample is detected using an assay system.

The characterization of an RSP virus is particularly desirable because it will allow for the determination of whether the virus is associated to the specific (restricted) or nonspecific (nonrestricted) pitting symptoms of RSP, or to both. Also, RSP virus resistant transgenic variants of the current commercial grape cultivars and rootstocks allows for more complete control of the virus while retaining the varietal characteristics of specifics cultivars. Furthermore, these variants permit control over RSP virus transmitted by infected scions or rootstocks. Moreover, the diagnostic tests offer significant improvement over conventional diagnostic means currently employed, namely, rapid results and greater accuracy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of St. George indicators which comparatively display the symptoms of RSP. The St. George indicator (a) has been graft-inoculated with infected bud wood from a grapevine accession, resulting in the indicator displaying pitting below the inoculum bud, as indicated by an arrow. This RSP symptom was defined by Goheen, “Rupestris Stem Pitting,” in Compendium of Grape Diseases, p. 53, Pearson and Goheen, eds., American Phytopathological Society Press, St. Paul, Minn., USA (1988), which is hereby incorporated by reference. The St. George indicator (b) was not graft-inoculated and represents a normal appearance.

FIGS. 2A and 2B are photographs which respectively display the results of dsRNA analysis and Northern hybridization for dsRNA. Together the photographs may be used to correlate the dsRNA analysis of FIG. 2A with the Northern hybridization (for dsRNA isolated from grapevines indexed positive for Rupestris stem pitting (RSP)) of FIG. 2B. M. Hind III digested lambda DNA maker: lane 1, Aminia; lane 2, Bertille Seyve 5563; lane 3, Canandaigua; lane 4, Colobel 257; lane 5, Couderc 28-112; lane 6, Freedom; lane 7, Grande Glabre; lane 8, M 344-1; lane 9, Joffre; lane 10, Ravat 34; lane 11, Seyval; lane 12, Seyve Vinard 14-287; lane 13, Verdelet; lane 14, Pinot Noir (positive control); lane 15, Verduzzo 233A (negative control for RSP as judged by indexing on St. George); lane 16, insert of clone RSP149. Arrows indicate the position of the 8.7 kb dsRNA. With respect to lane 15 of FIG. 2A, the two dsRNA bands are larger or smaller than the 8.7 kb dsRNA associated with RSP and they did not hybridize with the RSP specific probe in Northern analysis. Thus, they are not specific to RSP.

FIG. 3A is an illustration which depicts the strategy for obtaining the complete nucleotide sequence of RSPaV-1. The overlapping regions of the nucleotide sequences of the sequenced clones and RT-PCR-amplified cDNA fragments are as follows: 52-375 for RSPA/RSP28; 677-1474 for RSP28/RSP3; 3673-3766 for RSP3/RSPB; 40094320 for RSPB/RSP94; 5377-5750 for RSP94/RSPC; 5794-6537 for RSPC/RSP95; 6579-6771 for RSPC/RSP140; and 8193-8632 for RSP140/TA5. FIG. 3B is an illustration which comparatively depicts the genome structures of RSPaV-1, ASPV, PVM, and PVX. Boxes with the same patterns represent the comparable ORFS.

FIG. 4A is a comparative sequence listing of amino acid sequences of region 1 (aa 1-372) of RSPaV-1 ORF1 with the corresponding sequences of carlavirus PVM and ASPV. The methyltransferase motif is underlined. Capital letters indicate consensus residues. FIG. 4B is a comparative sequence listing of amino acid sequences of region II (aa 1354 to end) of RSPaV-1 ORF1 with the corresponding regions of ASPV and PVM carlavirus. In FIG. 4B, the NTP binding motif is underlined at (A) and the GDD containing sequence is underlined at (B). In FIGS. 4A and 4B, capital letters indicate consensus residues, the symbol * indicates identical amino acid residues between RSPaV-1 and ASPV, and the symbol # indicates identical amino acid residues between RSPaV-1 and PMV.

FIGS. 5A-D are comparative sequence listings of amino acid sequences for ORF2, ORF3, ORF74, and a C-terminal part of ORF5 (CP) of RSPaV-1, respectively, with ASPV and PVM carlavirus. In FIG. 5A, the NTP binding motif, located near the C terminus of ORF2, is underlined. In FIG. 5D, the conserved motif (RR/QX—XFDF), located in the central region of the coat proteins and proposed to be involved in the formation of a salt bridge structure, is underlined. In each of the figures, capital letters indicate consensus residues. The symbol * indicates identical amino acid residues between RSPaV-1 and ASPV, and the symbol # indicates identical amino acid residues between RSPaV-1 and PMV. In FIG. 5D, numbers which appear in parentheses and precede the sequences indicate the start points of the C-terminal portions of CPs being compared.

FIG. 6A is a comparative sequence listing of DNA nucleotide sequences for the 3′ untranslated region (UTR) of RSPaV-1 and ASPV. FIG. 6B is a comparative sequence listing of DNA nucleotide sequences for the 3′ untranslated region (UTR) of RSPaV-1 and PVM. Clustal method of MegAlign (DNASTAR) was used to generate sequence alignments. The 21 identical consecutive nucleotides between RSPaV-1 and PVM are indicated as shadowed letters.

FIGS. 7A-B are photographs comparing the results of RT-PCR of grapevines using RSP 149 primers (FIG. 7A) and Southern blot hybridization of RT-PCR amplified cDNA fragments to RSPaV-1 specific probe (FIG. 7B). MMLV-RT (Promega) was used in reverse transcription. Taq DNA polymerase (Promega) was used in PCR. For the RT-PCR and Southern blot hybridization: lane 1, Ehrenfelser PM1 (1169-1A1); lane 2, Cabernet franc 147A; lane 3, Chardonnay 80A; lane 4, Refosco 181A; lane 5, Touriga francesa 313; lane 6, 3309C (330-4A1); lane 7, 420A (1483-4A1); lane 8, Chardonnay 83A; lane 9, Malsavia 153A; lane 10, Aragnonex 350; lane 11, Aminia; lane 12, Chardonnay 127; lane 13, Kober 5BB 100; lane 14, Verduzzo 233A; lane 15, V. riparia; lane 16, V. monticola; lane 17, H₂O.

FIG. 8 is a schematic representation of the identical genome organization among RSPaV-1 (the type strain), RSP47-4, and RSP158. The number of amino acid residues of the comparable ORFs (boxes shaded with the same pattern) among these three strains are the same (note: ORFI and ORF5 of RSP47-4 and RSP158 are incomplete). The comparable ORFs also have high nucleotide and amino acid sequence identities, which are indicated on the bottom. Only the C-terminal portion of the ORF1 of RSPaV-1 is shown in this diagram.

FIG. 9 is a comparative alignment of nucleotide sequences of seven other clones with the comparable region of RSPaV-1. Shaded areas indicate identical nucleotide sequences, whereas white boxes represent different nucleotide sequences.

FIG. 10 is a schematic representation of a plant transformation vector containing the RSPaV-1 coat protein gene. This vector is designated pGA482G/RSPaV-1CP, which has the double CaMV 35S enhancers, the 35S promoter, the leader sequence of AIMV, and the 35S terminator sequence. RB, right border; LB, left border; Tet, tetracycline resistance gene; and Gent, gentamycin resistance gene.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated DNA molecules encoding for the proteins or polypeptides of a Rupestris stem pitting associated virus. Since the nucleotide sequence was derived from cDNA clones of the dsRNA that was associated with RSP, the viral agent has been designated as Rupestris stem pitting associated virus (“RSPaV”). RSP is likely caused by one or a number of viral strains. The genome of each RSPaV has a plurality of open reading frames, each containing DNA molecules in accordance with the present invention. The complete genome of one strain has been sequenced and the strain is designated RSPaV-1. Substantial portions of the genomes of two other RSPaV strains have also been sequenced. These strains are designated by their clone names, RSP47-4 and RSP158.

The DNA molecule which constitutes the complete RSPaV-1 genome comprises the nucleotide sequence corresponding to SEQ. ID. No. 1 as follows: CGATAAACAT AACAACAGAA TGTGCATTGC AGTAATATTC CTTGAATATA ATTGCAACGC   60 AATGGCCCTC TCTTATAGGC CTGCTGTTGA AGAGGTGCTC GCAAAATTCA CCTCTGATGA  120 ACAATCCAGG GTTTCTGCTA CAGCTCTCAA GGCATTAGTA GACTTAGAGG AAAGTCAGCA  180 CAATTTGTTC TCTTTCGCAT TGCCTGATAG AAGCAAAGAA AGGCTGATAT CTTCTGGCAT  240 TTACTTAAGT CCTTACAGTT TCAGACCCCA CTCACATCCA GTTTGTAAAA CTTTAGAAAA  300 TCACATTTTG TACAATGTTT TACCTAGTTA TGTTAATAAT TCATTTTACT TTGTAGGAAT  360 CAAGGATTTT AAGCTGCAGT TCTTGAAAAG GAGGAATAAG GATCTCAGCT TGGTAGCACT  420 CATAAATAGG TTTGTGACAA GTCGTGATGT TAGTAGGTAT GGGTCTGAGT TCGTTATAAG  480 TTCTAGTGAC AAATCAAGTC AGGTTGTCAG TAGAAAGGGC ATTGGTGATT CTAACACACT  540 CCGGAGATTG GTCCCACGTG TAATTTCCAC AGGTGCCAGG AATCTTTTTC TGCATGATGA  600 GATTCACTAC TGGTCAATTA GTGATCTGAT CAATTTTTTG GACGTTGCCA AGCCAAGCAT  660 GCTCTTGGCA ACTGCAGTAA TCCCTCCAGA AGTGCTGGTT GGCTCTCCAG AGAGTCTTAA  720 CCCTTGGGCC TACCAGTATA AAATCAATGG CAACCAACTG CTCTTCGCAC CAGATGGCAA  780 CTGGAATGAG ATGTACTCAC AACCTTTGTC ATGCAGATAC CTGCTCAAGG CCAGATCTGT  840 AGTTCTGCCC GATGGCTCAC GCTACTCGGT TGACATCATT CACTCAAAAT TTAGTCACCA  900 CTTGCTTAGT TTCACCCCTA TGGGTAATCT TTTGACTTCA AACATGCGAT GTTTTTCTGG  960 CTTCGATGCA ATAGGCATAA AAGATCTTGA ACCTCTAAGC CGCGGCATGC ACAGTTGCTT 1020 CCCAGTACAT CATGATGTTG TAACTAAGAT ATATCTTTAT TTGAGAACTC TCAAGAAGCC 1080 AGATAAGGAG TCTGCCGAGG CAAAGCTTCG ACAACTCATA GAAAAACCCA CAGGGAGGGA 1140 GATAAAGTTT ATCGAGGATT TTTCCTCACT AGTAATAAAT TGTGGGAGGA GTGGCTCTTT 1200 GCTTATGCCC AACATTTCTA AGTTGGTCAT ATCATTCTTT TGCCGGATGA TGCCAAATGC 1260 ACTCGCCAGG CTCTCTTCTA GCTTTCGAGA GTGTTCGCTA GATTCATTTG TGTACTCACT 1320 TGAGCCCTTT AATTTTTCCG TTAATTTAGT GGATATAACT CCTGATTTCT TTGAGCATTT 1380 ATTTCTCTTC TCCTGCCTAA ATGAGTTGAT CGAGGAGGAC GTTGAAGAGG TCATGGACAA 1440 TTCTTGGTTT GGACTTGGGG ACTTACAATT CAATCGCCAG AGGGCCCCGT TCTTTCTTGG 1500 GTCTTCATAT TGGCTCAACT CCAAATTTTC AGTTGAGCAC AAGTTTTCAG GCACCATCAA 1560 TTCTCAAATC ATGCAAGTTA TTTTATCTTT GATCCCATTT TCTGATGATC CCACTTTTAG 1620 GCCATCTTCT ACAGAGGTTA ACCTTGCACT ATCAGAGGTT AAGGCTGCGC TAGAAGCTAC 1680 TGGGCAGTCA AAATTGTTCA GGTTTTTGGT GGACGACTGT GCTATGCGTG AGGTTAGAAG 1740 TTCCTATAAG GTGGGCCTTT TTAAGCACAT AAAAGCCCTC ACTCATTGCT TTAATTCTTG 1800 TGGCCTCCAA TGGTTCCTCC TTAGGCAAAG GTCCAACCTC AAATTTCTGA AGGACAGGGC 1860 ATCGTCCTTT GCTGATCTTG ATTGTGAGGT TATCAAAGTT TATCAGCTTG TAACATCACA 1920 GGCAATACTT CCTGAGGCTC TGCTTAGCTT GACCAAAGTC TTTGTCAGGG ATTCTGACTC 1980 AAAGGGTGTT TCCATTCCCA GATTGGTCTC GAGAAATGAG CTAGAGGAAC TAGCTCACCC 2040 AGCTAATTCA GCCCTTGAGG AGCCTCAATC AGTTGATTGT AATGCAGGCA GGGTTCAAGC 2100 AAGCGTTTCA AGTTCCGAGC AGCTTGCCGA CACCCACTCT CTTGGTAGCG TTAAGTCATC 2160 AATTGAGACA GCTAACAAGG CTTTTAACTT GGAGGAGCTA AGGATCATGA TTAGAGTCTT 2220 GCCGGAGGAT TTTAACTGGG TGGCGAAGAA CATTGGTTTT AAAGACAGGC TGAGAGGCAG 2280 GGGTGCATCA TTCTTCTCAA AACCAGGAAT TTCATGTCAT AGTTACAATG GTGGGAGCCA 2340 CACAAGCTTA GGGTGGCCAA AGTTCATGGA TCAGATTCTA AGCTCCACTG GTGGACGTAA 2400 TTACTACAAT TCATGCCTGG CTCAGATCTA TGAGGAAAAT TCAAAATTGG CTCTTCATAA 2460 GGATGATGAG AGTTGCTATG AAATTGGGCA CAAAGTTTTG ACTGTTAATT TAATCGGCTC 2520 AGCAACTTTC ACTATTAGTA AGTCGCGAAA TTTGGTTGGG GGTAATCATT GCAGCCTGAC 2580 AATTGGGCCA AATGAGTTTT TCGAAATGCC TAGGGGCATG CAATGCAATT ACTTCCATGG 2640 GGTTTCCAAT TGTACGCCAG GGCGGGTATC GCTGACCTTT AGGCGCCAAA AGTTGGAAGA 2700 TGATGATTTG ATCTTCATAA ATCCACAGGT GCCCATTGAG CTCAATCATG AAAAGCTTGA 2760 CCGAAGTATG TGGCAGATGG GCCTTCATGG AATTAAGAAA TCTATTTCTA TGAATGGCAC 2820 GAGTTTTACC TCAGACCTAT GCTCTTGTTT CTCTTGCCAC AACTTTCATA AATTCAAGGA 2880 TCTCATCAAT AACTTGAGAT TGGCCCTAGG AGCACAAGGG CTAGGTCAGT GTGACAGGGT 2940 TGTGTTTGCA ACAACAGGTC CTGGTCTATC TAAGGTTTTA GAAATGCCTC GGAGCAAAAA 3000 GCAATCAATT TTGGTTCTTG AAGGTGCCCT ATCCATAGAA ACAGATTATG GTCCAAAAGT 3060 CCTGGGGTCT TTTGAAGTTT TCAAAGGGGA CTTTCACATT AAGAAGATGG AGGAAGGTTC 3120 AATTTTTGTA ATAACGTACA AGGCCCCAAT TAGATCCACT GGCAGGTTGA GGGTTCACAG 3180 TTCAGAATGC TCATTTTCCG GATCCAAAGA GGTATTGCTA GGCTGCCAGA TTGAGGCATG 3240 TGCTGATTAT GATATTGATG ATTTTAACAC TTTCTCTGTG CCTGGTGATG GCAATTGCTT 3300 TTGGCATTCT GTTGGTTTTT TACTTAGCAC TGATGGACTT GCCCTAAAGG CCGGTATTCG 3360 ATCTTTCGTG GAGAGTGAGC GCTTGGTAAG TCCAGATCTT TCAGCCCCAG CAATTTCTAA 3420 ACAATTGGAA GAGAATGCTT ATGCCGAGAA TGAGATGATC GCATTATTCT GCATTCGGCA 3480 CCACGTAAGG CCTATAGTGA TCACACCAGA ATATGAAGTT AGTTGGAAAT TCGGGGAAGG 3540 TGAGTGGCCC CTATGTGGAA TTCTTTGCCT TAAATCAAAT CACTTCCAAC CATGCGCCCC 3600 ACTGAATGGT TGCATGATCA CAGCCATTGC TTCAGCACTT GGAAGGCGTG AAGTTGATGT 3660 GTTAAATTAT CTGTGTAGAC CCAGCACTAA TCATATTTTT GAGGAGCTTT GTCAGGGAGG 3720 GGGCCTTAAC ATGATGTATT TAGCTGAAGC TTTTGAGGCC TTTGACATTT GCGCTAAATG 3780 TGATATAAAT GGAGAGATTG AAGTGATTAA TCCGTGTGGT AAAATTTCTG CATTGTTTGA 3840 CATAACTAAT GAGCACATAA GGCATGTTGA GAAAATAGGT AATGGCCCTC AGAGCATAAA 3900 AGTGGATGAA TTGCGGAAGG TCAAGCGATC CGCCCTCGAT TTCCTTTCAA TGAATGGGTC 3960 TAAAATAACC TACTTCCCAA GCTTTGAGCG GGCTGAAAAG TTGCAAGGAT GTTTGCTAGG 4020 GGGCCTAACT GGCGTTATAA GTGATGAGAA GTTCAGTGAT GCAAAACCTT GGCTTTCTGG 4080 TATATCTACT ACTGATATTA AGCCAAGGGA ATTGACTGTC GTGCTTGGTA CATTTGGGGC 4140 TGGGAAGAGT TTCTTGTACA AGAGTTTCAT GAAAAGGTCT GAGGGTAAAT TCGTAACCTT 4200 TGTTTCTCCC AGACGTGCTT TAGCAAATTC AATCAAAAAT GATCTTGAAA TGGATGATAG 4260 CTGCAAAGTT GCTAAAGCAG GTAGGTCAAA GAAGGAAGGG TGGGATGTAG TAACTTTTGA 4320 GGTTTTCCTT AGAAAAGTTG CAGGATTGAA GGCTGGCCAC TGTGTGATTT TTGATGAGGT 4380 CCAGTTGTTT CCTCCTGGAT ACATCGATCT ATGCTTGCTT ATTATACGTA GTGATGCTTT 4440 CATTTCACTT GCTGGTGATC CATGTCAAAG CACATATGAC TCGCAAAAGG ATCGGGCAAT 4500 TTTGGGCGCT GAGCAGAGTG ACATACTTAG ACTGCTTGAG GGCAAAACGT ATAGGTATAA 4560 CATAGAAAGC AGGAGGTTTG TGAACCCAAT GTTCGAATCA AGACTGCCAT GTCACTTCAA 4620 AAAGGGCTCG ATGACTGCCG CTTTCGCTGA TTATGCAATC TTCCATAATA TGCATGACTT 4680 TCTCCTGGCG AGGTCAAAAG GTCCCTTGGA TGCCGTTTTG GTTTCCAGTT TTGAGGAGAA 4740 AAAGATAGTC CAGTCCTACT TTGGAATGAA ACAGCTCACA CTCACATTTG GTGAATCAAC 4800 TGGGTTGAAT TTCAAAAATG GGGGAATTCT CATATCACAT GATTCCTTTC ACACAGATGA 4860 TCGGCGGTGG CTTACTGCTT TATCTCGCTT CAGCCACAAT TTGGATTTGG TGAACATCAC 4920 AGGTCTGAGG GTGGAAAGTT TTCTCTCGCA CTTTGCTGGC AAACCCCTCT ACCATTTTTT 4980 AACAGCCAAA AGTGGGGAGA ATGTCATACG AGATTTGCTC CCAGGTGAGC CTAACTTCTT 5040 CAGTGGCTTT AACGTTAGCA TTGGAAAGAA TGAAGGTGTT AGGGAGGAGA AGTTATGTGG 5100 TGACCCATGG TTAAAAGTTA TGCTTTTCCT GGGTCAAGAT GAGGATTGTG AAGTTGAAGA 5160 GATGGAGTCA GAATGCTCAA ATGAAGAATG GTTTAAAACC CACATCCCCT TGAGTAATCT 5220 GGAGTCAACC AGGGCCAGGT GGGTGGGTAA AATGGCCTTG AAAGAGTATC GGGAGGTGCG 5280 TTGTGGTTAT GAAATGACTC AACAATTCTT TGATGAGCAT AGGGGTGGAA CTGGTGAGCA 5340 ACTGAGCAAT GCATGTGAGA GGTTTGAAAG CATTTACCCA AGGCATAAAG GAAATGATTC 5400 AATAACCTTC CTCATGGCTG TCCGAAAGCG TCTCAAATTT TCGAAGCCCC AGGTTGAAGC 5460 TGCCAAACTG AGGCGGGCCA AACCATATGG GAAATTCTTA TTAGATTCTT TCCTATCCAA 5520 AATCCCATTG AAAGCCAGTC ATAATTCCAT CATGTTTCAT GAAGCGGTAC AGGAGTTTGA 5580 GGCGAAGAAG GCTAGTAAGA GTGCAGCAAC TATAGAGAAT CATGCAGGTA GGTCATGCAG 5640 GGATTGGTTA TTAGATGTTG CTCTGATTTT TATGAAGTCA CAACACTGTA CTAAATTTGA 5700 CAACAGGCTT AGAGTAGCTA AAGCTGGGCA AACCCTTGCT TGCTTCCAAC ATGCTGTTCT 5760 GGTTCGCTTT GCACCCTATA TGAGATACAT TGAGAAAAAG CTAATGCAAG CTCTGAAGCC 5820 TAACTTCTAC ATCCATTCAG GGAAAGGTCT GACGAGCTGA AGGAGTGGGT CAGAACTAGA 5880 GGATTCACTG GAATTTGCAC AGAATCAGAC TACGAAGCCT TTGATGCTTC CCAAGACCAC 5940 TTCATCCTAG CATTCGAATT GCAGATAATG AAATTTTTGG GGTTACCTGA AGATTTAATT 6000 TTGGACTATG AATTCATAAA AATTCATTTG GGATCAAAGC TCGGATCATT CTCTATAATG 6060 AGGTTTACTG GGGAGGCCAG CACATTTCTG TTTAACACTA TGGCTAACAT GTTGTTCACC 6120 TTTCTGAGGT ACGAACTAAC AGGCTCTGAG TCAATAGCAT TTGCAGGTGA TGACATGTGT 6180 GCTAATCGAA GGTTGCGGCT TAAAACAGAG CATGAGGGTT TTCTGAACAT GATTTGCCTT 6240 AAGGCCAAGG TTCAGTTTGT TTCCAATCCC ACATTCTGCG GATGGTGTTT ATTTAAGGAA 6300 GGGATCTTCA AGAAGCCTCA ATTAATCTGG GAGCGGATAT GCATTGCTAG GGAGATGGGC 6360 AACCTGGAGA ATTGTATTGA CAATTATGCG ATAGAGGTCT CCTATGCATA CCGACTGGGA 6420 GAGCTAGCCA TTGAAATGAT GACCGAGGAA GAAGTGGAGG CCCATTATAA TTGTGTTAGA 6480 TTCTTGGTCA GGAACAAGCA TAAGATGAGA TGCTCAATTT CAGGCCTATT TGAAGCTATT 6540 GATTAGGCCT TAAGTATTTG GCATTATTTG AGTATTATGA ATAATTTAGT TAAAGCATTG 6600 TCAGCATTTG AGTTTGTAGG TGTTTTCAGT GTGCTTAAAT TTCCAGTAGT CATTCATAGT 6660 GTGCCTGGTA GTGGTAAAAG TAGTTTAATA AGGGAGCTAA TTTCCGAGGA TGAGAATTTC 6720 ATAGCTTTCA CAGCAGGTGT TCCAGACAGC CCTAATCTCA CAGGAAGGTA CATTAAGCCT 6780 TATTCTCCAG GGTGTGCAGT GCCAGGGAAA GTTAATATAC TTGATGAGTA CTTGTCCGTC 6840 CAAGATTTTT CAGGTTTTGA TGTGCTGTTC TCGGACCCAT ACCAAAACAT CAGCATTCCT 6900 AAAGAGGCAC ATTTCATCAA GTCAAAAACT TGTAGGTTTG GCGTGAATAC TTGCAAATAT 6960 CTTTCCTCCT TCGGTTTTAA GGTTAGCAGT GACGGTTTGG ACAAAGTCAT TGTGGGGTCG 7020 CCTTTTACAC TAGATGTTGA AGGGGTGCTA ATATGCTTTG GTAAGGAGGC AGTGGATCTC 7080 GCTGTTGCGC ACAACTCTGA ATTCAAATTA CCTTGTGAAG TTAGAGGTTC AACTTTTAAC 7140 GTCGTAACTC TTTTGAAATC AAGAGATCCA ACCCCAGAGG ATAGGCACTG GTTTTACATT 7200 GCTGCTACAA GACACAGGGA GAAATTGATA ATCATGCAGT AAGATGCCTT TTCAGCAGCC 7260 TGCGAATTGG GCAAAAACCA TAACTCCATT GACAGTTGGC TTGGGCATTG GGCTTGTGCT 7320 GCATTTTCTG AGGAAGTCAA ATCTACCTTA TTCAGGGGAC AACATCCATC AATTCCCTCA 7380 CGGTGGGCGT TACAGGGACG GTACAAAAAG TATAACTTAC TGTGGTCCAA AGCAATCCTT 7440 CCCCAGCTCT GGGATATTCG GCCAATCTGA GAATTTTGTG CCCTTAATGC TTGTCATAGG 7500 TCTAATCGCA TTCATACATG TATTGTCTGT TTGGAATTCT GGTCTTGGTA GGAATTGTAA 7560 TTGCCATCCA AATCCTTGCT CATGTAGACA GCAGTAGTGG CAACCACCAA GGTTGCTTCA 7620 TTAGGGCCAC TGGAGAGTCA ATTTTGATTG AAAACTGCGG CCCAAGTGAG GCCCTTGCAT 7680 CCACTGTGAA GGAGGTGCTG GGAGGTTTGA AGGCTTTAGG GGTTAGCCGT GCTGTTGAAG 7740 AAATTGATTA TCATTGTTAA ATTGGCTGAA TGGCAAGTCA AATTGGGAAA CTCCCCGGTG 7800 AATCAAATGA GGCTTTTGAA GCCCGGCTAA AATCGCTGGA GTTAGCTAGA GCTCAAAAGC 7860 AGCCGGAAGG TTCTAATGCA CCACCTACTC TCAGTGGCAT TCTTGCCAAA CGCAAGAGGA 7920 TTATAGAGAA TGCACTTTCA AAGACGGTGG ACATGAGGGA GGTTTTGAAA CACGAAACGG 7980 TGGTGATTTC CCCAAATGTC ATGGATGAAG GTGCAATAGA CGAGCTGATT CGTGCATTTG 8040 GTGAATCTGG CATAGCTGAA AGCGTGCAAT TTGATGTGGC CATAGATATA GCACGTCACT 8100 GCTCTGATGT TGGTAGCTCC CAGAGGTCAA CCCTGATTGG CAAGAGTCCA TTTTGTGACC 8160 TAAACAGATC AGAAATAGCT GGGATTATAA GGGAGGTGAC CACATTACGT AGATTTTGCA 8220 TGTACTATGC AAAAATCGTG TGGAACATCC ATCTGGAGAC GGGGATACCA CCAGCTAACT 8280 GGGCCAAGAA AGGATTTAAT GAGAATGAAA AGTTTGCAGC CTTTGATTTT TTCTTGGGAG 8340 TCACAGATGA GAGTGCGCTT GAACCAAAGG GTGGAATTAA AAGAGCTCCA ACGAAAGCTG 8400 AGATGGTTGC TAATATCGCC TCTTTTGAGG TTCAAGTGCT CAGACAAGCT ATGGCTGAAG 8460 GCAAGCGGAG TTCCAACCTT GGAGAGATTA GTGGTGGAAC GGCTGGTGCA CTCATCAACA 8520 ACCCCTTTTC AAATGTTACA CATGAATGAG GATGACGAAG TCAGCGACAA TTCCGCAGTC 8580 CAATAATTCC CCGATTTCAA GGCTGGGTTA AGCCTGTTCG CTGGAATACC GTACTAATAG 8640 TATTCCCTTT CCATGCTAAA TCCTATTTAA TATATAAGGT GTGGAAAGTA AAAGAAGATT 8700 TGGTGTGTTT TTATAGTTTT CATTCAAAAA AAAAAAAAAA AAA 8743 The DNA molecule of SEQ. ID. No. 1 contains at least five open reading frames (e.g., ORF 1-ORF5), each of which encodes a particular protein or polypeptide of RSPaV-1, and a 3′ untranscribed region downstream of ORF5.

Another DNA molecule of the present invention (RSPaV-1 ORF 1) includes nucleotides 62-6547 of SEQ. ID. No. 1. The DNA molecule of RSPaV-1 ORF1 encodes for a RSPaV-1 replicase and comprises a nucleotide sequence corresponding to SEQ. ID. No. 2 as follows: ATGGCCCTCT CTTATAGGCC TGCTGTTGAA GAGGTGCTCG CAAAATTCAC CTCTGATGAA   60 CAATCCAGGG TTTCTGCTAC AGCTCTCAAG GCATTAGTAG ACTTAGAGGA AAGTCAGCAC  120 AATTTGTTCT CTTTCGCATT GCCTGATAGA AGCAAAGAAA GGCTGATATC TTCTGGCATT  180 TACTTAAGTC CTTACAGTTT CAGACCCCAC TCACATCCAG TTTGTAAAAC TTTAGAAAAT  240 CACATTTTGT ACAATGTTTT ACCTAGTTAT GTTAATAATT CATTTTACTT TGTAGGAATC  300 AAGGATTTTA AGCTGCAGTT CTTGAAAAGG AGGAATAAGG ATCTCAGCTT GGTAGCACTC  360 ATAAATAGGT TTGTGACAAG TCGTGATGTT AGTAGGTATG GGTCTGAGTT CGTTATAAGT  420 TCTAGTGACA AATCAAGTCA GGTTGTCAGT AGAAAGGGCA TTGGTGATTC TAACACACTC  480 CGGAGATTGG TCCCACGTGT AATTTCCACA GGTGCCAGGA ATCTTTTTCT GCATGATGAG  540 ATTCACTACT GGTCAATTAG TGATCTGATC AATTTTTTGG ACGTTGCCAA GCCAAGCATG  600 CTCTTGGCAA CTGCAGTAAT CCCTCCAGAA GTGCTGGTTG GCTCTCCAGA GAGTCTTAAC  660 CCTTGGGCCT ACCAGTATAA AATCAATGGC AACCAACTGC TCTTCGCACC AGATGGCAAC  720 TGGAATGAGA TGTACTCACA ACCTTTGTCA TGCAGATACC TGCTCAAGGC CAGATCTGTA  780 GTTCTGCCCG ATGGCTCACG CTACTCGGTT GACATCATTC ACTCAAAATT TAGTCACCAC  840 TTGCTTAGTT TCACCCCTAT GGGTAATCTT TTGACTTCAA ACATGCGATG TTTTTCTGGC  900 TTCGATGCAA TAGGCATAAA AGATCTTGAA CCTCTAAGCC GCGGCATGCA CAGTTGCTTC  960 CCAGTACATC ATGATGTTGT AACTAAGATA TATCTTTATT TGAGAACTCT CAAGAAGCCA 1020 GATAAGGAGT CTGCCGAGGC AAAGCTTCGA CAACTCATAG AAAAACCCAC AGGGAGGGAG 1080 ATAAAGTTTA TCGAGGATTT TTCCTCACTA GTAATAAATT GTGGGAGGAG TGGCTCTTTG 1140 CTTATGCCCA ACATTTCTAA GTTGGTCATA TCATTCTTTT GCCGGATGAT GCCAAATGCA 1200 CTCGCCAGGC TCTCTTCTAG CTTTCGAGAG TGTTCGCTAG ATTCATTTGT GTACTCACTT 1260 GAGCCCTTTA ATTTTTCCGT TAATTTAGTG GATATAACTC CTGATTTCTT TGAGCATTTA 1320 TTTCTCTTCT CCTGCCTAAA TGAGTTGATC GAGGAGGACG TTGAAGAGGT CATGGACAAT 1380 TCTTGGTTTG GACTTGGGGA CTTACAATTC AATCGCCAGA GGGCCCCGTT CTTTCTTGGG 1440 TCTTCATATT GGCTCAACTC CAAATTTTCA GTTGAGCACA AGTTTTCAGG CACCATCAAT 1500 TCTCAAATCA TGCAAGTTAT TTTATCTTTG ATCCCATTTT CTGATGATGC CACTTTTAGG 1560 CCATCTTCTA CAGAGGTTAA CCTTGCACTA TCAGAGGTTA AGGCTGCGCT AGAAGCTACT 1620 GGGCAGTCAA AATTGTTCAG GTTTTTGGTG GACGACTGTG CTATGCGTGA GGTTAGAAGT 1680 TCCTATAAGG TGGGCCTTTT TAAGCACATA AAAGCCCTCA CTCATTGCTT TAATTCTTGT 1740 GGCCTCCAAT GGTTCCTCCT TAGGCAAAGG TCCAACCTCA AATTTCTGAA GGACAGGGCA 1800 TCGTCCTTTG CTGATCTTGA TTGTGAGGTT ATCAAAGTTT ATCAGCTTGT AACATCACAG 1860 GCAATACTTC CTGAGGCTCT GCTTAGCTTG ACCAAAGTCT TTGTCAGGGA TTCTGACTCA 1920 AAGGGTGTTT CCATTCCCAG ATTGGTCTCG AGAAATGAGC TAGAGGAACT AGCTCACCCA 1980 GCTAATTCAG CCCTTGAGGA GCCTCAATCA GTTGATTGTA ATGCAGGCAG GGTTCAAGCA 2040 AGCGTTTCAA GTTCCCAGCA GCTTGCCGAC ACCCACTCTC TTGGTAGCGT TAAGTCATCA 2100 ATTGAGACAG CTAACAAGGC TTTTAACTTG GAGGAGCTAA GGATCATGAT TAGAGTCTTG 2160 CCGGAGGATT TTAACTGGGT GGCGAAGAAC ATTGGTTTTA AAGAGAGGCT GAGAGGCAGG 2220 GGTGCATCAT TCTTCTCAAA ACCAGGAATT TCATGTCATA GTTACAATGG TGGGAGCCAC 2280 ACAAGCTTAG GGTGGCCAAA GTTCATGGAT CAGATTCTAA GCTCCACTGG TGGACGTAAT 2340 TACTACAATT CATGCCTGGC TCAGATCTAT GAGGAAAATT CAAAATTGGC TCTTCATAAG 2400 GATGATGAGA GTTGCTATGA AATTGGGCAC AAAGTTTTGA CTGTTAATTT AATCGGCTCA 2460 GCAACTTTCA CTATTAGTAA GTCGCGAAAT TTGGTTGGGG GTAATCATTG CAGCCTGACA 2520 ATTGGGCCAA ATGAGTTTTT CGAAATGCCT AGGGGCATGC AATGCAATTA CTTCCATGGG 2580 GTTTCCAATT GTACGCCAGG GCGGGTATCG CTGACCTTTA GGCGCCAAAA GTTGGAAGAT 2640 GATGATTTGA TCTTCATAAA TCCACAGGTG CCCATTGAGC TCAATCATGA AAAGCTTGAC 2700 CGAAGTATGT GGCAGATGGG CCTTCATGGA ATTAAGAAAT CTATTTCTAT GAATGGCACG 2760 AGTTTTACCT CAGACCTATG CTCTTGTTTC TCTTGCCACA ACTTTCATAA ATTCAAGGAT 2820 CTCATCAATA ACTTGAGATT GGCCCTAGGA GCACAAGGGC TAGGTCAGTG TGACAGGGTT 2880 GTGTTTGCAA CAACAGGTCC TGGTCTATCT AAGGTTTTAG AAATGCCTCG GAGCAAAAAG 2940 CAATCAATTT TGGTTCTTGA AGGTGCCCTA TCCATAGAAA CAGATTATGG TCCAAAAGTC 3000 CTGGGGTCTT TTGAAGTTTT CAAAGGGGAC TTTCACATTA AGAAGATGGA GGAAGGTTCA 3060 ATTTTTGTAA TAACGTACAA GGCCCCAATT AGATCCACTG GCAGGTTGAG GGTTCACAGT 3120 TCAGAATGCT CATTTTCCGG ATCCAAAGAG GTATTGCTAG GCTGCCAGAT TGAGGCATGT 3180 GCTGATTATG ATATTGATGA TTTTAACACT TTCTCTGTGC CTGGTGATGG CAATTGCTTT 3240 TGGCATTCTG TTGGTTTTTT ACTTAGCACT GATGGACTTG CCGTAAAGGC CGGTATTCGA 3300 TCTTTCGTGG AGAGTGAGCG CTTGGTAAGT CCAGATCTTT CAGCCCCAGC AATTTCTAAA 3360 CAATTGGAAG AGAATGCTTA TGCCGAGAAT GAGATGATCG CATTATTCTG CATTCGGCAC 3420 CACGTAAGGC CTATAGTGAT CACACCAGAA TATGAAGTTA GTTGGAAATT CGGGGAAGGT 3480 GAGTGGCCCC TATGTGGAAT TCTTTGCCTT AAATCAAATC ACTTCCAAGC ATGCGCCCCA 3540 CTGAATGGTT GCATGATCAC AGCCATTGCT TCAGCAGTTG GAAGGCGTGA AGTTGATGTG 3600 TTAAATTATC TGTGTAGACC GAGCACTAAT CATATTTTTG AGGAGGTTTG TCAGGGAGGG 3660 GGCCTTAACA TGATGTATTT AGCTGAAGCT TTTGAGGGCT TTGACATTTG CGGTAAATGT 3720 GATATAAATG GAGAGATTGA AGTGATTAAT CCGTGTGGTA AAATTTCTGC ATTGTTTGAC 3780 ATAACTAATG AGCACATAAG GCATGTTGAG AAAATAGGTA ATGGCCCTCA GAGCATAAAA 3840 GTGGATGAAT TGCGGAAGGT CAAGCGATCC GCGCTCGATT TCCTTTCAAT GAATGGGTCT 3900 AAAATAACCT ACTTCCCAAG CTTTGAGCGG GCTGAAAAGT TGCAAGGATG TTTGCTAGGG 3960 GGCCTAACTG GCGTTATAAG TGATGAGAAG TTCAGTGATG CAAAACCTTG GCTTTCTGGT 4020 ATATCTACTA CTGATATTAA GCCAAGGGAA TTGACTGTCG TGCTTGGTAC ATTTGGGGCT 4080 GGGAAGAGTT TCTTGTACAA GAGTTTCATG AAAAGGTCTG AGGGTAAATT CGTAACCTTT 4140 GTTTCTCCCA GACGTGCTTT AGCAAATTCA ATCAAAAATG ATCTTGAAAT GGATGATAGC 4200 TGCAAAGTTG CTAAAGCAGG TAGGTCAAAG AAGGAAGGGT GGGATGTAGT AACTTTTGAG 4260 GTTTTCCTTA GAAAAGTTGC AGGATTGAAG GCTGGCCACT GTGTGATTTT TGATGAGGTC 4320 CAGTTGTTTC CTCCTGGATA CATCGATCTA TGCTTGCTTA TTATACGTAG TGATGCTTTC 4380 ATTTCACTTG CTGGTGATCC ATGTCAAAGC ACATATGACT CGCAAAAGGA TCGGGCAATT 4440 TTGGGCGCTG AGCAGAGTGA CATACTTAGA CTGCTTGAGG GCAAAACGTA TAGGTATAAC 4500 ATAGAAAGCA GGAGGTTTGT GAACCCAATG TTCGAATCAA GACTGCCATG TCACTTCAAA 4560 AAGGGCTCGA TGACTGCCGC TTTCGCTGAT TATGCAATCT TCCATAATAT GCATGACTTT 4620 CTCCTGGCGA GGTCAAAAGG TCCCTTGGAT GCCGTTTTGG TTTCCAGTTT TGAGGAGAAA 4680 AAGATAGTCC AGTCGTACTT TGGAATGAAA CAGCTCACAC TCACATTTGG TGAATCAACT 4740 GGGTTGAATT TCAAAAATGG GGGAATTCTC ATATCACATG ATTCCTTTCA CACAGATGAT 4800 CGGCGGTGGC TTACTGCTTT ATCTCGCTTC AGCCACAATT TGGATTTGGT GAACATCACA 4860 GGTCTGAGGG TGGAAAGTTT TCTCTCGCAC TTTGCTGGCA AACCCCTCTA CCATTTTTTA 4920 ACAGCCAAAA GTGGGGAGAA TGTCATACGA GATTTGCTCC CAGGTGAGCC TAACTTCTTC 4980 AGTGGCTTTA ACGTTAGCAT TGGAAAGAAT GAAGGTGTTA GGGAGGAGAA GTTATGTGGT 5040 GACCCATGGT TAAAAGTTAT GCTTTTGCTG GGTCAAGATG AGGATTGTGA AGTTGAAGAG 5100 ATGGAGTCAG AATGCTCAAA TGAAGAATGG TTTAAAACCC ACATCCGCTT GAGTAATCTG 5160 GAGTCAACCA GGGCCAGGTG GGTGGGTAAA ATGGCCTTGA AAGAGTATCG GGAGGTGCGT 5220 TGTGGTTATG AAATGACTCA ACAATTCTTT GATGAGCATA GGGGTGGAAC TGGTGAGCAA 5280 CTGAGCAATG CATGTGAGAG GTTTGAAAGC ATTTACCCAA GGCATAAAGG AAATGATTCA 5340 ATAACCTTCC TCATGGGTGT CCGAAAGCGT CTCAAATTTT CGAAGCCCCA GGTTGAAGCT 5400 GCCAAACTGA GGCGGGCCAA AGCATATGGG AAATTCTTAT TAGATTCTTT CCTATCCAAA 5460 ATCCCATTGA AAGCCAGTCA TAATTCCATC ATGTTTCATG AAGCGGTACA GGAGTTTGAG 5520 GCGAAGAAGG CTAGTAAGAG TGCAGCAACT ATAGAGAATC ATGCAGGTAG GTCATGCAGG 5580 GATTGGTTAT TAGATGTTGG TCTGATTTTT ATGAAGTCAC AACACTGTAC TAAATTTGAC 5640 AACAGGCTTA GAGTAGCTAA AGCTGGGCAA ACCCTTGCTT GCTTCCAACA TGCTGTTCTG 5700 GTTCGCTTTG CACCCTATAT GAGATACATT GAGAAAAAGC TAATGCAAGC TCTGAAGCCT 5760 AACTTCTACA TCCATTCAGG GAAAGGTGTG ACGAGCTGAA CGAGTGGGTC AGAACTAGAG 5820 GATTCACTGG AATTTGCACA GAATCAGACT ACGAAGCCTT TGATGCTTCC CAAGACCACT 5880 TCATCCTAGC ATTCGAATTG CAGATAATGA AATTTTTGGG GTTACCTGAA GATTTAATTT 5940 TGGACTATGA ATTCATAAAA ATTCATTTGG GATCAAAGCT CGGATCATTC TCTATAATGA 6000 GGTTTACTGG GGAGGCCAGC ACATTTCTGT TTAACACTAT GGCTAACATG TTGTTCACCT 6060 TTCTGAGGTA CGAACTAACA GGCTCTGAGT CAATAGGATT TGCAGGTGAT GACATGTGTG 6120 CTAATCGAAG GTTGCGGCTT AAAACAGAGC ATGAGGGTTT TCTGAACATG ATTTGCCTTA 6180 AGGCCAAGGT TCAGTTTGTT TCCAATCCCA CATTCTGCGG ATGGTGTTTA TTTAAGGAAG 6240 GGATCTTCAA GAAGCCTCAA TTAATCTGGG AGCGGATATG CATTGCTAGG GAGATGGGCA 6300 ACCTGGAGAA TTGTATTGAC AATTATGCGA TAGAGGTCTC CTATGCATAC CGACTGGGAG 6360 AGCTAGCCAT TGAAATGATG ACCGAGGAAG AAGTGGAGGC CCATTATAAT TGTGTTAGAT 6420 TCTTGGTCAG GAACAAGCAT AAGATGAGAT GCTCAATTTC AGGCCTATTT GAAGCTATTG 6480 ATTAG 6485

The RSPaV-1 replicase has a deduced amino acid sequence corresponding to SEQ. ID. No. 3 as follows: Met Ala Leu Ser Tyr Arg Pro Ala Val Glu Glu Val 1               5                   10 Leu Ala Lys Phe Thr Ser Asp Glu Gln Ser Arg Val         15                  20 Ser Ala Thr Ala Leu Lys Ala Leu Val Asp Leu Glu 25                  30                  35 Glu Ser Gln His Asn Leu Phe Ser Phe Ala Leu Pro             40                  45 Asp Arg Ser Lys Glu Arg Leu Ile Ser Ser Gly Ile     50                  55                  60 Tyr Leu Ser Pro Tyr Ser Phe Arg Pro His Ser His                 65                  70 Pro Val Cys Lys Thr Leu Glu Asn His Ile Leu Tyr         75                  80 Asn Val Leu Pro Ser Tyr Val Asn Asn Ser Phe Tyr 85                  90                  95 Phe Val Gly Ile Lys Asp Phe Lys Leu Gln Phe Leu             100                 105 Lys Arg Arg Asn Lys Asp Leu Ser Leu Val Ala Leu     110                 115                 120 Ile Asn Arg Phe Val Thr Ser Arg Asp Val Ser Arg                 125                 130 Tyr Gly Ser Glu Phe Val Ile Ser Ser Ser Asp Lys         135                 140 Ser Ser Gln Val Val Ser Arg Lys Gly Ile Gly Asp 145                 150                 155 Ser Asn Thr Leu Arg Arg Leu Val Pro Arg Val Ile             160                 165 Ser Thr Gly Ala Arg Asn Leu Phe Leu His Asp Glu     170                 175                 180 Ile His Tyr Trp Ser Ile Ser Asp Leu Ile Asn Phe                 185                 190 Leu Asp Val Ala Lys Pro Ser Met Leu Leu Ala Thr         195                 200 Ala Val Ile Pro Pro Glu Val Leu Val Gly Ser Pro 205                 210                 215 Glu Ser Leu Asn Pro Trp Ala Tyr Gln Tyr Lys Ile             220                 225 Asn Gly Asn Gln Leu Leu Phe Ala Pro Asp Gly Asn     230                 235                 240 Trp Asn Glu Met Tyr Ser Gln Pro Leu Ser Cys Arg                 245                 250 Tyr Leu Leu Lys Ala Arg Ser Val Val Leu Pro Asp         255                 260 Gly Ser Arg Tyr Ser Val Asp Ile Ile His Ser Lys 265                 270                 275 Phe Ser His His Leu Leu Ser Phe Thr Pro Met Gly             280                 285 Asn Leu Leu Thr Ser Asn Met Arg Cys Phe Ser Gly     290                 295                 300 Phe Asp Ala Ile Gly Ile Lys Asp Leu Glu Pro Leu                 305                 310 Ser Arg Gly Met His Ser Cys Phe Pro Val His His         315                 320 Asp Val Val Thr Lys Ile Tyr Leu Tyr Leu Arg Thr 325                 330                 335 Leu Lys Lys Pro Asp Lys Glu Ser Ala Glu Ala Lys             340                 345 Leu Arg Gln Leu Ile Glu Lys Pro Thr Gly Arg Glu     350                 355                 360 Ile Lys Phe Ile Glu Asp Phe Ser Ser Leu Val Ile                 365                 370 Asn Cys Gly Arg Ser Gly Ser Leu Leu Met Pro Asn         375                 380 Ile Ser Lys Leu Val Ile Ser Phe Phe Cys Arg Met 385                 390                 395 Met Pro Asn Ala Leu Ala Arg Leu Ser Ser Ser Phe             400                 405 Arg Glu Cys Ser Leu Asp Ser Phe Val Tyr Ser Leu     410                 415                 420 Glu Pro Phe Asn Phe Ser Val Asn Leu Val Asp Ile                 425                 430 Thr Pro Asp Phe Phe Glu His Leu Phe Leu Phe Ser         435                 440 Cys Leu Asn Glu Leu Ile Glu Glu Asp Val Glu Glu 445                 450                 455 Val Met Asp Asn Ser Trp Phe Gly Leu Gly Asp Leu             460                 465 Gln Phe Asn Arg Gln Arg Ala Pro Phe Phe Leu Gly     470                 475                 480 Ser Ser Tyr Trp Leu Asn Ser Lys Phe Ser Val Glu                 485                 490 His Lys Phe Ser Gly Thr Ile Asn Ser Gln Ile Met         495                 500 Gln Val Ile Leu Ser Leu Ile Pro Phe Ser Asp Asp 505                 510                 515 Pro Thr Phe Arg Pro Ser Ser Thr Glu Val Asn Leu             520                 525 Ala Leu Ser Glu Val Lys Ala Ala Leu Glu Ala Thr     530                 535                 540 Gly Gln Ser Lys Leu Phe Arg Phe Leu Val Asp Asp                 545                 550 Cys Ala Met Arg Glu Val Arg Ser Ser Tyr Lys Val         555                 560 Gly Leu Phe Lys His Ile Lys Ala Leu Thr His Cys 565                 570                 575 Phe Asn Ser Cys Gly Leu Gln Trp Phe Leu Leu Arg             580                 585 Gln Arg Ser Asn Leu Lys Phe Leu Lys Asp Arg Ala     590                 595                 600 Ser Ser Phe Ala Asp Leu Asp Cys Glu Val Ile Lys                 605                 610 Val Tyr Gln Leu Val Thr Ser Gln Ala Ile Leu Pro         615                 620 Glu Ala Leu Leu Ser Leu Thr Lys Val Phe Val Arg 625                 630                 635 Asp Ser Asp Ser Lys Gly Val Ser Ile Pro Arg Leu             640                 645 Val Ser Arg Asn Glu Leu Glu Glu Leu Ala His Pro     650                 655                 660 Ala Asn Ser Ala Leu Glu Glu Pro Gln Ser Val Asp                 665                 670 Cys Asn Ala Gly Arg Val Gln Ala Ser Val Ser Ser         675                 680 Ser Gln Gln Leu Ala Asp Thr His Ser Leu Gly Ser 685                 690                 695 Val Lys Ser Ser Ile Glu Thr Ala Asn Lys Ala Phe             700                 705 Asn Leu Glu Glu Leu Arg Ile Met Ile Arg Val Leu     710                 715                 720 Pro Glu Asp Phe Asn Trp Val Ala Lys Asn Ile Gly                 725                 730 Phe Lys Asp Arg Leu Arg Gly Arg Gly Ala Ser Phe         735                 740 Phe Ser Lys Pro Gly Ile Ser Cys His Ser Tyr Asn 745                 750                 755 Gly Gly Ser His Thr Ser Leu Gly Trp Pro Lys Phe             760                 765 Met Asp Gln Ile Leu Ser Ser Thr Gly Gly Arg Asn     770                 775                 780 Tyr Tyr Asn Ser Cys Leu Ala Gln Ile Tyr Glu Glu                 785                 790 Asn Ser Lys Leu Ala Leu His Lys Asp Asp Glu Ser         795                 800 Cys Tyr Glu Ile Gly His Lys Val Leu Thr Val Asn 805                 810                 815 Leu Ile Gly Ser Ala Thr Phe Thr Ile Ser Lys Ser             820                 825 Arg Asn Leu Val Gly Gly Asn His Cys Ser Leu Thr     830                 835                 840 Ile Gly Pro Asn Glu Phe Phe Glu Met Pro Arg Gly                 845                 850 Met Gln Cys Asn Tyr Phe His Gly Val Ser Asn Cys         855                 860 Thr Pro Gly Arg Val Ser Leu Thr Phe Arg Arg Gln 865                 870                 875 Lys Leu Glu Asp Asp Asp Leu Ile Phe Ile Asn Pro             880                 885 Gln Val Pro Ile Glu Leu Asn His Glu Lys Leu Asp     890                 895                 900 Arg Ser Met Trp Gln Met Gly Leu His Gly Ile Lys                 905                 910 Lys Ser Ile Ser Met Asn Gly Thr Ser Phe Thr Ser         915                 920 Asp Leu Cys Ser Cys Phe Ser Cys His Asn Phe His 925                 930                 935 Lys Phe Lys Asp Leu Ile Asn Asn Leu Arg Leu Ala             940                 945 Leu Gly Ala Gln Gly Leu Gly Gln Cys Asp Arg Val     950                 955                 960 Val Phe Ala Thr Thr Gly Pro Gly Leu Ser Lys Val                 965                 970 Leu Glu Met Pro Arg Ser Lys Lys Gln Ser Ile Leu         975                 980 Val Leu Glu Gly Ala Leu Ser Ile Glu Thr Asp Tyr 985                 990                 995 Gly Pro Lys Val Leu Gly Ser Phe Glu Val Phe Lys             1000                1005 Gly Asp Phe His Ile Lys Lys Met Glu Glu Gly Ser     1010                1015                1020 Ile Phe Val Ile Thr Tyr Lys Ala Pro Ile Arg Ser                 1025                1030 Thr Gly Arg Leu Arg Val His Ser Ser Glu Cys Ser         1035                1040 Phe Ser Gly Ser Lys Glu Val Leu Leu Gly Cys Gln 1045                1050                1055 Ile Glu Ala Cys Ala Asp Tyr Asp Ile Asp Asp Phe             1060                1065 Asn Thr Phe Ser Val Pro Gly Asp Gly Asn Cys Phe     1070                1075                1080 Trp His Ser Val Gly Phe Leu Leu Ser Thr Asp Gly                 1085                1090 Leu Ala Leu Lys Ala Gly Ile Arg Ser Phe Val Glu         1095                1100 Ser Glu Arg Leu Val Ser Pro Asp Leu Ser Ala Pro 1105                1110                1115 Ala Ile Ser Lys Gln Leu Glu Glu Asn Ala Tyr Ala             1120                1125 Glu Asn Glu Met Ile Ala Leu Phe Cys Ile Arg His     1130                1135                1140 His Val Arg Pro Ile Val Ile Thr Pro Glu Tyr Glu                 1145                1150 Val Ser Trp Lys Phe Gly Glu Gly Glu Trp Pro Leu         1155                1160 Cys Gly Ile Leu Cys Leu Lys Ser Asn His Phe Gln 1165                1170                1175 Pro Cys Ala Pro Leu Asn Gly Cys Met Ile Thr Ala             1180                1185 Ile Ala Ser Ala Leu Gly Arg Arg Glu Val Asp Val     1190                1195                1200 Leu Asn Tyr Leu Cys Arg Pro Ser Thr Asn His Ile                 1205                1210 Phe Glu Glu Leu Cys Gln Gly Gly Gly Leu Asn Met         1215                1220 Met Tyr Leu Ala Glu Ala Phe Glu Ala Phe Asp Ile 1225                1230                1235 Cys Ala Lys Cys Asp Ile Asn Gly Glu Ile Glu Val             1240                1245 Ile Asn Pro Cys Gly Lys Ile Ser Ala Leu Phe Asp     1250                1255                1260 Ile Thr Asn Glu His Ile Arg His Val Glu Lys Ile                 1265                1270 Gly Asn Gly Pro Gln Ser Ile Lys Val Asp Glu Leu         1275                1280 Arg Lys Val Lys Arg Ser Ala Leu Asp Phe Leu Ser 1285                1290                1295 Met Asn Gly Ser Lys Ile Thr Tyr Phe Pro Ser Phe             1300                1305 Glu Arg Ala Glu Lys Leu Gln Gly Cys Leu Leu Gly     1310                1315                1320 Gly Leu Thr Gly Val Ile Ser Asp Glu Lys Phe Ser                 1325                1330 Asp Ala Lys Pro Trp Leu Ser Gly Ile Ser Thr Thr         1335                1340 Asp Ile Lys Pro Arg Glu Leu Thr Val Val Leu Gly 1345                1350                1355 Thr Phe Gly Ala Gly Lys Ser Phe Leu Tyr Lys Ser             1360                1365 Phe Met Lys Arg Ser Glu Gly Lys Phe Val Thr Phe     1370                1375                1380 Val Ser Pro Arg Arg Ala Leu Ala Asn Ser Ile Lys                 1385                1390 Asn Asp Leu Glu Met Asp Asp Ser Cys Lys Val Ala         1395                1400 Lys Ala Gly Arg Ser Lys Lys Glu Gly Trp Asp Val 1405                1410                1415 Val Thr Phe Glu Val Phe Leu Arg Lys Val Ala Gly             1420                1425 Leu Lys Ala Gly His Cys Val Ile Phe Asp Glu Val     1430                1435                1440 Gln Leu Phe Pro Pro Gly Tyr Ile Asp Leu Cys Leu                 1445                1450 Leu Ile Ile Arg Ser Asp Ala Phe Ile Ser Leu Ala         1455                1460 Gly Asp Pro Cys Gln Ser Thr Tyr Asp Ser Gln Lys 1465                1470                1475 Asp Arg Ala Ile Leu Gly Ala Glu Gln Ser Asp Ile             1480                1485 Leu Arg Leu Leu Glu Gly Lys Thr Tyr Arg Tyr Asn     1490                1495                1500 Ile Glu Ser Arg Arg Phe Val Asn Pro Met Phe Glu                 1505                1510 Ser Arg Leu Pro Cys His Phe Lys Lys Gly Ser Met         1515                1520 Thr Ala Ala Phe Ala Asp Tyr Ala Ile Phe His Asn 1525                1530                1535 Met His Asp Phe Leu Leu Ala Arg Ser Lys Gly Pro             1540                1545 Leu Asp Ala Val Leu Val Ser Ser Phe Glu Glu Lys     1550                1555               1560 Lys Ile Val Gln Ser Tyr Phe Gly Met Lys Gln Leu                 1565                1570 Thr Leu Thr Phe Gly Glu Ser Thr Gly Leu Asn Phe         1575                1580 Lys Asn Gly Gly Ile Leu Ile Ser His Asp Ser Phe 1585                1590                1595 His Thr Asp Asp Arg Arg Trp Leu Thr Ala Leu Ser             1600                1605 Arg Phe Ser His Asn Leu Asp Leu Val Asn Ile Thr     1610                1615                1620 Gly Leu Arg Val Glu Ser Phe Leu Ser His Phe Ala                 1625                1630 Gly Lys Pro Leu Tyr His Phe Leu Thr Ala Lys Ser         1635                1640 Gly Glu Asn Val Ile Arg Asp Leu Leu Pro Gly Glu 1645                1650                1655 Pro Asn Phe Phe Ser Gly Phe Asn Val Ser Ile Gly             1660                1665 Lys Asn Glu Gly Val Arg Glu Glu Lys Leu Cys Gly     1670                1675                1680 Asp Pro Trp Leu Lys Val Met Leu Phe Leu Gly Gln                 1685                1690 Asp Glu Asp Cys Glu Val Glu Glu Met Glu Ser Glu         1695                1700 Cys Ser Asn Glu Glu Trp Phe Lys Thr His Ile Pro 1705                1710                1715 Leu Ser Asn Leu Glu Ser Thr Arg Ala Arg Trp Val             1720                1725 Gly Lys Met Ala Leu Lys Glu Tyr Arg Glu Val Arg     1730                1735                1740 Cys Gly Tyr Glu Met Thr Gln Gln Phe Phe Asp Glu                 1745                1750 His Arg Gly Gly Thr Gly Glu Gln Leu Ser Asn Ala         1755                1760 Cys Glu Arg Phe Glu Ser Ile Tyr Pro Arg His Lys 1765                1770                1775 Gly Asn Asp Ser Ile Thr Phe Leu Met Ala Val Arg             1780                1785 Lys Arg Leu Lys Phe Ser Lys Pro Gln Val Glu Ala     1790                1795                1800 Ala Lys Leu Arg Arg Ala Lys Pro Tyr Gly Lys Phe                 1805                1810 Leu Leu Asp Ser Phe Leu Ser Lys Ile Pro Leu Lys         1815                1820 Ala Ser His Asn Ser Ile Met Phe His Glu Ala Val 1825                1830                1835 Gln Glu Phe Glu Ala Lys Lys Ala Ser Lys Ser Ala             1840                1845 Ala Thr Ile Glu Asn His Ala Gly Arg Ser Cys Arg     1850                1855                1860 Asp Trp Leu Leu Asp Val Ala Leu Ile Phe Met Lys                 1865                1870 Ser Gln His Cys Thr Lys Phe Asp Asn Arg Leu Arg        1875                 1880 Val Ala Lys Ala Gly Gln Thr Leu Ala Cys Phe Gln 1885                1890                1895 His Ala Val Leu Val Arg Phe Ala Pro Tyr Met Arg             1900                1905 Tyr Ile Glu Lys Lys Leu Met Gln Ala Leu Lys Pro     1910                1915                1920 Asn Phe Tyr Ile His Ser Gly Lys Gly Leu Asp Glu                 1925                1930 Leu Asn Glu Trp Val Arg Thr Arg Gly Phe Thr Gly         1935                1940 Ile Cys Thr Glu Ser Asp Tyr Glu Ala Phe Asp Ala 1945                1950                1955 Ser Gln Asp His Phe Ile Leu Ala Phe Glu Leu Gln             1960                1965 Ile Met Lys Phe Leu Gly Leu Pro Glu Asp Leu Ile     1970                1975                1980 Leu Asp Tyr Glu Phe Ile Lys Ile His Leu Gly Ser                 1985                1990 Lys Leu Gly Ser Phe Ser Ile Met Arg Phe Thr Gly         1995                2000 Glu Ala Ser Thr Phe Leu Phe Asn Thr Met Ala Asn 2005                2010                2015 Met Leu Phe Thr Phe Leu Arg Tyr Glu Leu Thr Gly             2020                2025 Ser Glu Ser Ile Ala Phe Ala Gly Asp Asp Met Cys     2030                2035                2040 Ala Asn Arg Arg Leu Arg Leu Lys Thr Glu His Glu                 2045                2050 Gly Phe Leu Asn Met Ile Cys Leu Lys Ala Lys Val         2055                2060 Gln Phe Val Ser Asn Pro Thr Phe Cys Gly Trp Cys 2065                2070                2075 Leu Phe Lys Glu Gly Ile Phe Lys Lys Pro Gln Leu             2080                2085 Ile Trp Glu Arg Ile Cys Ile Ala Arg Glu Met Gly     2090                2095                2100 Asn Leu Glu Asn Cys Ile Asp Asn Tyr Ala Ile Glu                 2105                2110 Val Ser Tyr Ala Tyr Arg Leu Gly Glu Leu Ala Ile         2115                2120 Glu Met Met Thr Glu Glu Glu Val Glu Ala His Tyr 2125                2130                2135 Asn Cys Val Arg Phe Leu Val Arg Asn Lys His Lys             2140                2145 Met Arg Cys Ser Ile Ser Gly Leu Phe Glu Ala Ile     2150                2155                2160 Asp The replicase of SEQ. ID. No. 3 has a molecular weight of about 240 to 246 kDa, preferably about 244 kDa.

Another DNA molecule of the present invention (RSPaV-1 ORF2) includes nucleotides 6578-7243 of SEQ. ID. No. 1. The DNA molecule of RSPaV-1 ORF2 encodes for a first protein or polypeptide of an RSPaV-1 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 4 as follows: ATGAATAATT TAGTTAAAGC ATTGTCAGCA TTTGAGTTTG TAGGTGTTTT CAGTGTGCTT  60 AAATTTCCAG TAGTCATTCA TAGTGTGCCT GGTAGTGGTA AAAGTAGTTT AATAAGGGAG 120 CTAATTTCCG AGGATGAGAA TTTCATAGCT TTCACAGCAG GTGTTCCAGA CAGCCCTAAT 180 CTCACAGGAA GGTACATTAA GCCTTATTCT CCAGGGTGTG CAGTGCCAGG GAAAGTTAAT 240 ATACTTGATG AGTACTTGTC CGTCCAAGAT TTTTCAGGTT TTGATGTGCT GTTCTCGGAC 300 CCATACCAAA ACATCAGCAT TCCTAAAGAG GCACATTTCA TCAAGTCAAA AACTTGTAGG 360 TTTGGCGTGA ATACTTGCAA ATATCTTTCC TCCTTCGGTT TTAAGGTTAG CAGTGACGGT 420 TTGGACAAAG TCATTGTGGG GTCGCCTTTT ACACTAGATG TTGAAGGGGT GCTAATATGC 480 TTTGGTAAGG AGGCAGTGGA TCTCGCTGTT GCGCACAACT CTGAATTCAA ATTACCTTGT 540 GAAGTTAGAG GTTCAACTTT TAACGTCGTA ACTCTTTTGA AATCAAGAGA TCCAACCCCA 600 GAGGATAGGC ACTGGTTTTA CATTGCTGCT ACAAGACACA GGGAGAAATT GATAATCATG 660 CAG 663

The first protein or polypeptide of the RSPaV-1 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 5 as follows: Met Asn Asn Leu Val Lys Ala Leu Ser Ala Phe Glu 1               5                   10 Phe Val Gly Val Phe Ser Val Leu Lys Phe Pro Val         15                  20 Val Ile His Ser Val Pro Gly Ser Gly Lys Ser Ser 25                  30                  35 Leu Ile Arg Glu Leu Ile Ser Glu Asp Glu Asn Phe             40                  45 Ile Ala Phe Thr Ala Gly Val Pro Asp Ser Pro Asn     50                  55                  60 Leu Thr Gly Arg Tyr Ile Lys Pro Tyr Ser Pro Gly                 65                  70 Cys Ala Val Pro Gly Lys Val Asn Ile Leu Asp Glu         75                  80 Tyr Leu Ser Val Gln Asp Phe Ser Gly Phe Asp Val 85                  90                  95 Leu Phe Ser Asp Pro Tyr Gln Asn Ile Ser Ile Pro             100                 105 Lys Glu Ala His Phe Ile Lys Ser Lys Thr Cys Arg     110                 115                 120 Phe Gly Val Asn Thr Cys Lys Tyr Leu Ser Ser Phe                 125                 130 Gly Phe Lys Val Ser Ser Asp Gly Leu Asp Lys Val         135                 140 Ile Val Gly Ser Pro Phe Thr Leu Asp Val Glu Gly 145                 150                 155 Val Leu Ile Cys Phe Gly Lys Glu Ala Val Asp Leu             160                 165 Ala Val Ala His Asn Ser Glu Phe Lys Leu Pro Cys     170                 175                 180 Glu Val Arg Gly Ser Thr Phe Asn Val Val Thr Leu                 185                 190 Leu Lys Ser Arg Asp Pro Thr Pro Glu Asp Arg His         195                 200 Trp Phe Tyr Ile Ala Ala Thr Arg His Arg Glu Lys 205                 210                 215 Leu Ile Ile Met Gln             220 The first protein or polypeptide of the RSPaV-1 triple gene block has a molecular weight of about 20 to 26 kDa, preferably 24.4 kDa.

Another DNA molecule of the present invention (RSPaV-1 ORF3) includes nucleotides 7245-7598 of SEQ. ID. No. 1. The DNA molecule of RSPaV-1 ORF3 encodes for a second protein or polypeptide of the triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 6 as follows: ATGCCTTTTC AGCAGCCTGC GAATTGGGCA AAAACCATAA CTCCATTGAC AGTTGGCTTG  60 GGCATTGGGC TTGTGCTGCA TTTTCTGAGG AAGTCAAATG TACCTTATTC AGGGGACAAC 120 ATCCATCAAT TCCCTCACGG TGGGCGTTAC AGGGACGGTA CAAAAAGTAT AACTTACTGT 180 GGTCCAAAGC AATCCTTCCC CAGCTCTGGG ATATTCGGCC AATCTGAGAA TTTTGTGCCC 240 TTAATGCTTG TCATAGGTCT AATCGCATTC ATACATGTAT TGTCTGTTTG GAATTCTGGT 300 CTTGGTAGGA ATTGTAATTG CCATCCAAAT CCTTGCTCAT GTAGACAGCA G 351

The second protein or polypeptide of the RSPaV-1 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 7 as follows: Met Pro Phe Gln Gln Pro Ala Asn Trp Ala Lys Thr 1               5                   10 Ile Thr Pro Leu Thr Val Gly Leu Gly Ile Gly Leu         15                  20 Val Leu His Phe Leu Arg Lys Ser Asn Leu Pro Tyr 25                  30                  35 Ser Gly Asp Asn Ile His Gln Phe Pro His Gly Gly             40                  45 Arg Tyr Arg Asp Gly Thr Lys Ser Ile Thr Tyr Cys     50                  55                  60 Gly Pro Lys Gln Ser Phe Pro Ser Ser Gly Ile Phe                 65                  70 Gly Gln Ser Glu Asn Phe Val Pro Leu Met Leu Val         75                  80 Ile Gly Leu Ile Ala Phe Ile His Val Leu Ser Val 85                  90                  95 Trp Asn Ser Gly Leu Gly Arg Asn Cys Asn Cys His             100                 105 Pro Asn Pro Cys Ser Cys Arg Gln Gln     110                 115

The second protein or polypeptide of the RSPaV-1 triple gene block has a molecular weight of about 10 to 15 kDa, preferably 12.8 kDa.

Yet another DNA molecule of the present invention (RSPaV-1 ORF4) includes nucleotides 7519-7761 of SEQ. ID. No. 1. The DNA molecule of RSPaV-1 ORF4 encodes for a third protein or polypeptide of the RSPaV-1 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 8 as follows: ATGTATTGTC TGTTTGGAAT TCTGGTCTTG GTAGGAATTG TAATTGCCAT CCAAATCCTT  60 GCTCATGTAG ACAGCAGTAG TGGCAACCAC CAAGGTTGCT TCATTAGGGC CACTGGAGAG 120 TCAATTTTGA TTGAAAACTG CGGCCCAAGT GAGGCCCTTG CATCCACTGT GAAGGAGGTG 180 CTGGGAGGTT TGAAGGCTTT AGGGGTTAGC CGTGCTGTTG AAGAAATTGA TTATCATTGT 240

The third protein or polypeptide of the RSPaV-1 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 9 as follows: Met Tyr Cys Leu Phe Gly Ile Leu Val Leu Val Gly 1               5                   10 Ile Val Ile Ala Ile Gln Ile Leu Ala His Val Asp         15                  20 Ser Ser Ser Gly Asn His Gln Gly Cys Phe Ile Arg 25                  30                  35 Ala Thr Gly Glu Ser Ile Leu Ile Glu Asn Cys Gly             40                  45 Pro Ser Glu Ala Leu Ala Ser Thr Val Lys Glu Val     50                  55                  60 Leu Gly Gly Leu Lys Ala Leu Gly Val Ser Arg Ala                 65                  70 Val Glu Glu Ile Asp Tyr His Cys         75                  80 The third protein or polypeptide of the RSPaV-1 triple gene block has a molecular weight of about 5 to 10 kDa, preferably 8.4 kDa.

Still another DNA molecule of the present invention (RSPaV-1 ORF5) includes nucleotides 7771-8550 of SEQ. ID. No. 1. The DNA molecule of RSPaV-1 ORF5 encodes for a RSPaV-1 coat protein and comprises a nucleotide sequence corresponding to SEQ. ID. No. 10 as follows: ATGGCAAGTC AAATTGGGAA ACTCCCCGGT GAATCAAATG AGGCTTTTGA AGCCCGGCTA  60 AAATCGCTGG AGTTAGCTAG AGCTCAAAAG CAGCCGGAAG GTTCTAATGC ACCACCTACT 120 CTCAGTGGCA TTCTTGCCAA ACGCAAGAGG ATTATAGAGA ATGGACTTTC AAAGACGGTG 180 GACATGAGGG AGGTTTTGAA ACACGAAACG GTGGTGATTT CCCCAAATGT CATGGATGAA 240 GGTGCAATAG ACGAGCTGAT TCGTGCATTT GGTGAATCTG GCATAGCTGA AAGCGTGCAA 300 TTTGATGTGG CCATAGATAT AGCACGTCAC TGCTCTGATG TTGGTAGCTC CCAGAGTTCA 360 ACCCTGATTG GCAAGAGTCC ATTTTGTGAC CTAAACAGAT CAGAAATAGC TGGGATTATA 420 AGGGAGGTGA CCACATTACG TAGATTTTGC ATGTACTATG CAAAAATCGT GTGGAACATC 480 CATCTGGAGA CGGGGATACC ACCAGCTAAC TGGGCCAAGA AAGGATTTAA TGAGAATGAA 540 AAGTTTGCAG CCTTTGATTT TTTCTTGGGA GTCACAGATG AGAGTGCGCT TGAACCAAAG 600 GGTGGAATTA AAAGAGCTCC AACGAAAGCT GAGATGGTTG CTAATATCGC CTCTTTTGAG 660 GTTCAAGTGC TCAGACAAGC TATGGCTGAA GGCAAGCGGA GTTCCAACCT TGGAGAGATT 720 AGTGGTGGAA CGGCTGGTGC ACTCATCAAC AACCCCTTTT CAAATGTTAC ACATGAA 777

The RSPaV-1 coat protein has a deduced amino acid sequence corresponding to SEQ. ID. No. 11 as follows: Met Ala Ser Gln Ile Gly Lys Leu Pro Gly Glu Ser Asn Glu Ala Phe 1               5                   10                  15 Glu Ala Arg Leu Lys Ser Leu Glu Leu Ala Arg Ala Gln Lys Gln Pro             20                  25                  30 Glu Gly Ser Asn Ala Pro Pro Thr Leu Ser Gly Ile Leu Ala Lys Arg         35                  40                  45 Lys Arg Ile Ile Glu Asn Ala Leu Ser Lys Thr Val Asp Met Arg Glu     50                  55                  60 Val Leu Lys His Glu Thr Val Val Ile Ser Pro Asn Val Met Asp Glu 65                  70                  75                  80 Gly Ala Ile Asp Glu Leu Ile Arg Ala Phe Gly Glu Ser Gly Ile Ala                 85                  90                  95 Glu Ser Val Gln Phe Asp Val Ala Ile Asp Ile Ala Arg His Cys Ser             100                 105                 110 Asp Val Gly Ser Ser Gln Ser Ser Thr Leu Ile Gly Lys Ser Pro Phe         115                 120                 125 Cys Asp Leu Asn Arg Ser Glu Ile Ala Gly Ile Ile Arg Glu Val Thr     130                 135                 140 Thr Leu Arg Arg Phe Cys Met Tyr Tyr Ala Lys Ile Val Trp Asn Ile 145                 150                 155                 160 His Leu Glu Thr Gly Ile Pro Pro Ala Asn Trp Ala Lys Lys Gly Phe                 165                 170                 175 Asn Glu Asn Glu Lys Phe Ala Ala Phe Asp Phe Phe Leu Gly Val Thr             180                 185                 190 Asp Glu Ser Ala Leu Glu Pro Lys Gly Gly Ile Lys Arg Ala Pro Thr         195                 200                 205 Lys Ala Glu Met Val Ala Asn Ile Ala Ser Phe Glu Val Gln Val Leu     210                 215                 220 Arg Gln Ala Met Ala Glu Gly Lys Arg Ser Ser Asn Leu Gly Glu Ile 225                 230                 235                 240 Ser Gly Gly Thr Ala Gly Ala Leu Ile Asn Asn Pro Phe Ser Asn Val                 245                 250                 255 Thr His Glu The RSPaV-1 coat protein has a molecular weight of about 25 to 30 kDa, preferably 28 kDa.

The DNA molecule which constitutes the substantial portion of the RSPaV strain RSP474 genome comprises the nucleotide sequence corresponding to SEQ. ID. No. 12 as follows: GGCTGGGCAA ACTTTGGCCT GCTTTCAACA CGCCGTCTTG GTTCGCTTTG CACCCTACAT 60 GCGATACATT GAAAAGAAGC TTGTGCAGGC ATTGAAACCA AATTTCTACA TTCATTCTGG 120 CAAAGGTCTT GATGAGCTAA GTGAATGGGT TAGAGCCAGA GGTTTCACAG GTGTGTGTAC 180 TGAGTCAGAC TATGAAGCTT TTGATGCATC CCAAGATCAT TTCATCCTGG CATTTGAACT 240 GCAAATCATG AGATTTTTAG GACTGCCAGA AGATCTGATT TTAGATTATG AGTTCATCAA 300 AATTCATCTT GGGTCAAAGC TTGGCTCTTT TGCAATTATG AGATTCACAG GTGAGGCAAG 360 CACCTTCCTA TTCAATACTA TGGCCAACAT GCTATTCACT TTCCTGAGGT ATGAGTTGAC 420 AGGTTCTGAA TCAATTGCAT TTGCTGGAGA TGATATGTGT GCTAATCGCA GGTTAAGACT 480 CAAGACTGAG CACGCCGGCT TTCTAAACAT GATCTGTCTC AAAGCTAAGG TGCAGTTTGT 540 CACAAATCCC ACCTTCTGTG GATGGTGTTT GTTTAAAGAG GGAATCTTTA AAAAACCCCA 600 GCTCATTTGG GAAAGGATCT GCATTGCTAG GGAAATGGGT AACTTGGACA ATTGCATTGA 660 CAATTACGCA ATTGAGGTGT CTTATGCTTA CAGACTTGGG GAATTGTCCA TAGGCGTGAT 720 GACTGAGGAG GAAGTTGAAG CACATTCTAA CTGCGTGCGT TTCCTGGTTC GCAATAAGCA 780 CAAGATGAGG TGCTCAATTT CTGGTTTGTT TGAAGTAATT GTTTAGGCCT TAAGTGTTTG 840 GCATGGTGTG AGTATTATGA ATAACTTAGT CAAAGCTTTG TCTGCTTTTG AATTTGTTGG 900 TGTGTTTTGT GTACTTAAAT TTCCAGTTGT TGTTCACAGT GTTCCAGGTA GCGGTAAAAG 960 TAGCCTAATA AGGGAGCTCA TTTCTGAAGA CGAGGCTTTT GTGGCCTTTA CAGCAGGTGT 1020 GCCAGACAGT CCAAATCTGA CAGGGAGGTA CATCAAGCCC TACGCTCCAG GGTGTGCAGT 1080 GCAAGGGAAA ATAAACATAC TTGATGAGTA GTTGTCTGTC TCTGATACTT CTGGCTTTGA 1140 TGTGCTGTTC TGAGACCCTT ACCAGAATGT CAGCATTCCA AGGGAGGCAC ACTTCATAAA 1200 AACCAAAACC TGTAGGTTTG GTACCAACAC CTGCAAGTAC CTTCAATCTT TTGGCTTTAA 1260 TGTTTGTAGT GATGGGGTGG ATAAAGTTGT TGTAGGGTCG CCATTTGAAC TGGAGGTTGA 1320 GGGGGTTCTC ATTTGCTTTG GAAAGGAGGC TGTAGATCTA GCAGTTGCAG ACAATTCTGA 1380 CTTCAAGTTG CCCTGCGAGG TGCGGGGTTC AAGATTTGAC GTTGTAACGT TATTGAAGTC 1440 CAGGGATCCA ACTTCAGAAG ATAAGCATTG GTTGTACGTT GCAGCCACAA GGCATCGAAG 1500 TAAACTGATA ATAATGCAGT AAAATGCCTT TTCAGCAACC TGCCAACTGG GCTAAGACCA 1560 TAACTCCATT AACTATTGGT TTGGGCATTG GGTTGGTTCT GCACTTCTTA AGGAAATCAA 1620 ATCTGCCATA TTCAGGAGAC AATATTCACC AGTTCCCACA CGGAGGGCAT TACAGGGACG 1680 GCACGAAGAG TATAACCTAT TGTGGCCCTA GGCAGTCATT CCCAAGCTCA GGAATATTCG 1740 GTCAGTCTGA AAATTTCGTA CCTCTAATAT TGGTCGTGAC TCTGGTCGCT TTTATACATG 1800 CGTTATCTCT TTGGAATTCT GGTCCTAGTA GGAGTTGCAA TTGCCATCCA AATCCTTGCA 1860 CATGTAGACA GCAGTAGTGG CAACCATCAA GGCTGTTTCA TAAGAGCCAC CGGGGAGTCA 1920 ATAGTAATTG AGAATTGTGG GCCGAGCGAG GCCCTAGCTG CTACAGTCAA AGAGGTGTTG 1980 GGCGGTCTAA AGGCTTTAGG GGTTAGCCAA AAGGTTGATG AAATTAATTA CAGTTGTTGA 2040 GACAGTTGAA TGGCAAGTCA AGTTGGAAAA TTGCCTGGCG AATCAAATGA AGCATATGAG 2100 GCTAGACTCA AGGCTTTAGA GTTAGCAAGG GCCCAAAAAG CTCCAGAAGT CTCCAACCAA 2160 CCTCCCACAC TTGGAGGCAT TCTAGCCAAA AGGAAAAGAG TGATTGAGAA TGCACTCTCA 2220 AAGACAGTGG ATATGCGTGA AGTCTTAAGG CATGAATCTG TTGTACTCTC CCCGAATGTA 2280 ATGGACGAGG GAGCAATAGA CGAGCTGATT GGTGCCTTTG GGGAGTCGGG CATAGCTGAA 2340 AATGTGCAGT TTGATGTTGC AATAGACATT GCTCGCCACT GTTCTGATGT GGGGAGCTCT 2400 CAGAGGTCAA CCCTTATTGG TAAAAGCCCC TTCTGTGAGT TAAATAGGTC TGAAATTGCC 2460 GGAATAATAA GGGAGGTGAC CACGCTGCGC AGATTTTGCA TGTACTACGC AAAGATTGTG 2520 TGGAACATCC ATTTGGAGAC GGGAATACCA GCAGCTAATT GGGCCAAGAA AGGATTTAAT 2580 GAGAATGAAA AGTTTGCAGC CTTTGACTTC TTCCTTGGAG TCACAGATGA AAGCGCGCTT 2640 GAGCCTAAGG GTGGAGTCAA GAGAGCTCCA ACAAAAGCAG 2680 The RSP47-4 strain contains five open reading frames (i.e., ORF1-5). ORF1 and ORF5 are only partially sequenced. RSP47-4 is 79% identical in nucleotide sequence to the corresponding region of RSPaV-1. The amino acid sequence identities between the corresponding ORFs of RSP47-4 and RSPaV-1 are: 94.1% for ORF1, 88.2% for ORF2, 88.9% for ORF3, 86.2% for ORF4, and 92.9% for ORF5. The nucleotide sequences of the five potential ORFs of RSP47-4 are given below.

Another DNA molecule of the present invention (RSP47-4 incomplete ORF1) includes nucleotides 1-768 of SEQ. ID. No. 12. This DNA molecule is believed to code for a polypeptide portion of a RSP47-4 replicase and comprises a nucleotide sequence corresponding to SEQ. ID. No. 13 as follows: ATGCGATACA TTGAAAAGAA GCTTGTGCAG GCATTGAAAC CAAATTTCTA CATTCATTCT 60 GGCAAAGGTC TTGATGAGCT AAGTGAATGG GTTAGAGGCA GAGGTTTCAC AGGTGTGTGT 120 ACTGAGTCAG ACTATGAAGC TTTTGATGCA TCCCAAGATC ATTTCATCCT GGCATTTGAA 180 CTGCAAATCA TGAGATTTTT AGGACTGCCA GAAGATCTGA TTTTAGATTA TGAGTTCATC 240 AAAATTCATC TTGGGTCAAA GCTTGGCTCT TTTGCAATTA TGAGATTCAC AGGTGAGGCA 300 AGCACCTTCG TATTCAATAC TATGGCCAAC ATGCTATTCA CTTTCCTGAG GTATGAGTTG 360 ACAGGTTCTG AATCAATTGC ATTTGCTGGA GATGATATGT GTGCTAATCG CAGGTTAAGA 420 CTCAAGACTG AGCACGCCGG CTTTCTAAAC ATGATCTGTC TCAAAGCTAA GGTGCAGTTT 480 GTCACAAATC CCACCTTCTG TGGATGGTGT TTGTTTAAAG AGGGAATCTT TAAAAAACCC 540 CAGCTCATTT GGGAAAGGAT CTGCATTGCT AGGGAAATGG GTAACTTGGA CAATTGCATT 600 GACAATTACG CAATTGAGGT GTCTTATGCT TACAGACTTG GGGAATTGTC CATAGGCGTG 660 ATGACTGAGG AGGAAGTTGA AGCACATTCT AACTGCGTGC GTTTCCTGGT TCGCAATAAG 720 CACAAGATGA GGTGCTCAAT TTCTGGTTTG TTTGAAGTAA TTGTTTA 767

The polypeptide has a deduced amino acid sequence corresponding to SEQ. ID. No. 14 as follows: Met Arg Tyr Ile Glu Lys Lys Leu Val Gln Ala Leu Lys Pro Asn Phe 1               5                   10                  15 Tyr Ile His Ser Gly Lys Gly Leu Asp Glu Leu Ser Glu Trp Val Arg             20                  25                  30 Ala Arg Gly Phe Thr Gly Val Cys Thr Glu Ser Asp Tyr Glu Ala Phe         35                  40                  45 Asp Ala Ser Gln Asp His Phe Ile Leu Ala Phe Glu Leu Gln Ile Met     50                  55                  60 Arg Phe Leu Gly Leu Pro Glu Asp Leu Ile Leu Asp Tyr Glu Phe Ile 65                  70                  75                  80 Lys Ile His Leu Gly Ser Lys Leu Gly Ser Phe Ala Ile Met Arg Phe                 85                  90                  95 Thr Gly Glu Ala Ser Thr Phe Leu Phe Asn Thr Met Ala Asn Met Leu             100                 105                 110 Phe Thr Phe Leu Arg Tyr Glu Leu Thr Gly Ser Glu Ser Ile Ala Phe         115                 120                 125 Ala Gly Asp Asp Met Cys Ala Asn Arg Arg Leu Arg Leu Lys Thr Glu     130                 135                 140 His Ala Gly Phe Leu Asn Met Ile Cys Leu Lys Ala Lys Val Gln Phe 145                 150                 155                 160 Val Thr Asn Pro Thr Phe Cys Gly Trp Cys Leu Phe Lys Glu Gly Ile                 165                 170                 175 Phe Lys Lys Pro Gln Leu Ile Trp Glu Arg Ile Cys Ile Ala Arg Glu             180                 185                 190 Met Gly Asn Leu Asp Asn Cys Ile Asp Asn Tyr Ala Ile Glu Val Ser         195                 200                 205 Tyr Ala Tyr Arg Leu Gly Glu Leu Ser Ile Gly Val Met Thr Glu Glu     210                 215                 220 Glu Val Glu Ala His Ser Asn Cys Val Arg Phe Leu Val Arg Asn Lys 225                 230                 235                 240 His Lys Met Arg Cys Ser Ile Ser Gly Leu Phe Glu Val Ile Val                 245                 250                 255

Another DNA molecule of the present invention (RSP47-4 ORF2) includes nucleotides 857-1522 of SEQ. ID. No. 12. This DNA molecule codes for a first protein or polypeptide of an RSP47-4 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 15 as follows: ATGAATAACT TAGTCAAAGC TTTGTCTGCT TTTGAATTTG TTGGTGTGTT TTGTGTACTT 60 AAATTTCCAG TTGTTGTTCA CAGTGTTCCA GGTAGCGGTA AAAGTAGCCT AATAAGGGAG 120 CTCATTTCTG AAGACGAGGC TTTTGTGGCC TTTACAGCAG GTGTGCCAGA CAGTCCAAAT 180 CTGACAGGGA GGTACATCAA GCCCTACGCT CCAGGGTGTG CAGTGCAAGG GAAAATAAAC 240 ATACTTGATG AGTACTTGTC TGTCTCTGAT ACTTCTGGCT TTGATGTGCT GTTCTCAGAC 300 CCTTACCAGA ATGTCAGCAT TCCAAGGGAG GCACACTTCA TAAAAACCAA AACCTGTAGG 360 TTTGGTACCA ACACCTGCAA GTACCTTCAA TCTTTTGGCT TTAATGTTTG TAGTGATGGG 420 GTGGATAAAG TTGTTGTAGG GTCGCCATTT GAACTGGAGG TTGAGGGGGT TCTCATTTGC 480 TTTGGAAAGG AGGCTGTAGA TCTAGCAGTT GCACACAATT CTGACTTCAA GTTGCCCTGC 540 GAGGTGCGGG GTTCAACATT TGACGTTGTA ACGTTATTGA AGTCCAGGGA TCCAACTTCA 600 GAAGATAAGC ATTGGTTCTA CGTTGCAGCC ACAAGGCATC GAAGTAAACT GATAATAATG 660 CAGTAA 666

The first protein or polypeptide of the RSP47-4 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 16 as follows: Met Asn Asn Leu Val Lys Ala Leu Ser Ala Phe Glu Phe Val Gly Val 1               5                   10                  15 Phe Cys Val Leu Lys Phe Pro Val Val Val His Ser Val Pro Gly Ser             20                  25                  30 Gly Lys Ser Ser Leu Ile Arg Glu Leu Ile Ser Glu Asp Glu Ala Phe         35                  40                  45 Val Ala Phe Thr Ala Gly Val Pro Asp Ser Pro Asn Leu Thr Gly Arg     50                  55                  60 Tyr Ile Lys Pro Tyr Ala Pro Gly Cys Ala Val Gln Gly Lys Ile Asn 65                  70                  75                  80 Ile Leu Asp Glu Tyr Leu Ser Val Ser Asp Thr Ser Gly Phe Asp Val                 85                  90                  95 Leu Phe Ser Asp Pro Tyr Gln Asn Val Ser Ile Pro Arg Glu Ala His             100                 105                 110 Phe Ile Lys Thr Lys Thr Cys Arg Phe Gly Thr Asn Thr Cys Lys Tyr         115                 120                 125 Leu Gln Ser Phe Gly Phe Asn Val Cys Ser Asp Gly Val Asp Lys Val     130                 135                 140 Val Val Gly Ser Pro Phe Glu Leu Glu Val Glu Gly Val Leu Ile Cys 145                 150                 155                 160 Phe Gly Lys Glu Ala Val Asp Leu Ala Val Ala His Asn Ser Asp Phe                 165                 170                 175 Lys Leu Pro Cys Glu Val Arg Gly Ser Thr Phe Asp Val Val Thr Leu             180                 185                 190 Leu Lys Ser Arg Asp Pro Thr Ser Glu Asp Lys His Trp Phe Tyr Val         195                 200                 205 Ala Ala Thr Arg His Arg Ser Lys Leu Ile Ile Met Gln     210                 215                 220 The first protein or polypeptide of the RSP47-4 triple gene block has a molecular weight of about 20 to 26 kDa., preferably 24.3 kDa.

Another DNA molecule of the present invention (RSP47-4 ORF3) includes nucleotides 1524-1877 of SEQ. ID. No. 12. This DNA molecule codes for a second protein or polypeptide of the RSP47-4 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 17 as follows: ATGCCTTTTG AGCAACCTGC CAACTGGGCT AAGACCATAA CTCCATTAAC TATTGGTTTG 60 GGCATTGGGT TGGTTCTGCA CTTCTTAAGG AAATCAAATC TGCCATATTC AGGAGACAAT 120 ATTCACCAGT TCCCACACGG AGGGCATTAC AGGGACGGCA CGAAGAGTAT AACCTATTGT 180 GGCCCTAGGC AGTCATTCCC AAGCTCAGGA ATATTCGGTC AGTCTGAAAA TTTCGTACCT 240 CTAATATTGG TCGTGACTCT GGTCGCTTTT ATACATGCGT TATCTCTTTG GAATTCTGGT 300 CCTAGTAGGA GTTGCAATTG CCATCCAAAT CCTTGCACAT GTAGACAGCA GTAG 354

The second protein or polypeptide of the RSP47-4 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 18 as follows: Met Pro Phe Gln Gln Pro Ala Asn Trp Ala Lys Thr Ile Thr Pro Leu 1               5                   10                  15 Thr Ile Gly Leu Gly Ile Gly Leu Val Leu His Phe Leu Arg Lys Ser             20                  25                  30 Asn Leu Pro Tyr Ser Gly Asp Asn Ile His Gln Phe Pro His Gly Gly         35                  40                  45 His Tyr Arg Asp Gly Thr Lys Ser Ile Thr Tyr Cys Gly Pro Arg Gln     50                  55                  60 Ser Phe Pro Ser Ser Gly Ile Phe Gly Gln Ser Glu Asn Phe Val Pro 65                  70                  75                  80 Leu Ile Leu Val Val Thr Leu Val Ala Phe Ile His Ala Leu Ser Leu                 85                  90                  95 Trp Asn Ser Gly Pro Ser Arg Ser Cys Asn Cys His Pro Asn Pro Cys             100                 105                 110 Thr Cys Arg Gln Gln         115 The second protein or polypeptide of the RSP47-4 triple gene block has a molecular weight of about 10 to 15 kDa., preferably 12.9 kDa.

Another DNA molecule of the present invention (RSP47-4 ORF4) includes nucleotides 1798-2040 of SEQ. ID. No. 12. This DNA molecule codes for a third protein or polypeptide of the RSP47-4 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 19 as follows: ATGCGTTATC TCTTTGGAAT TCTGGTCCTA GTAGGAGTTG CAATTGCCAT CCAAATCCTT 60 GCACATGTAG ACAGCAGTAG TGGCAACCAT CAAGGCTGTT TCATAAGAGC CACCGGGGAG 120 TCAATAGTAA TTGAGAATTG TGGGCCGAGC GAGGCCCTAG CTGCTACAGT CAAAGAGGTG 180 TTGGGCGGTC TAAAGGCTTT AGGGGTTAGC CAAAAGGTTG ATGAAATTAA TTACAGTTGT 240 TGA 243

The third protein or polypeptide of the RSP47-4 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 20 as follows: Met Arg Tyr Leu Phe Gly Ile Leu Val Leu Val Gly Val Ala Ile Ala 1               5                   10                  15 Ile Gln Ile Leu Ala His Val Asp Ser Ser Ser Gly Asn His Gln Gly             20                  25                  30 Cys Phe Ile Arg Ala Thr Gly Glu Ser Ile Val Ile Glu Asn Cys Gly         35                  40                  45 Pro Ser Glu Ala Leu Ala Ala Thr Val Lys Glu Val Leu Gly Gly Leu     50                  55                  60 Lys Ala Leu Gly Val Ser Gln Lys Val Asp Glu Ile Asn Tyr Ser Cys 65                  70                  75                  80 The third protein or polypeptide of the RSP47-4 triple gene block has a molecular weight of about 5 to 10 kDa., preferably 8.3 kDa.

Yet another DNA molecule of the present invention (RSP474 ORF5) includes nucleotides 2050-2680 of SEQ. ID. No. 12. This DNA molecule codes for a partial RSP47-4 coat protein or polypeptide and comprises a nucleotide sequence corresponding to SEQ. ID. No. 21 as follows: ATGGCAAGTC AAGTTGGAAA ATTGCCTGGC GAATCAAATG AAGCATATGA GGCTAGACTC 60 AAGGCTTTAG AGTTAGCAAG GGCCCAAAAA GCTCCAGAAG TCTCCAACCA ACCTCCCACA 120 CTTGGAGGCA TTCTAGCCAA AAGGAAAAGA GTGATTGAGA ATGCACTCTC AAAGACAGTG 180 GATATGCGTG AAGTCTTAAG GCATGAATCT GTTGTACTCT CCCCGAATGT AATGGACGAG 240 GGAGCAATAG ACGAGCTGAT TCGTGCCTTT GGGGAGTCGG GCATAGCTGA AAATGTGCAG 300 TTTGATGTTG CAATAGACAT TGCTCGCCAC TGTTCTGATG TGGGGAGCTC TCAGAGGTCA 360 ACCGTTATTG GTAAAAGCCC CTTCTGTGAG TTAAATAGGT CTGAAATTGC CGGAATAATA 420 AGGGAGGTGA CCACGCTGCG CAGATTTTGC ATGTACTACG CAAAGATTGT GTGGAACATC 480 CATTTGGAGA CGGGAATACC ACCAGCTAAT TGGGCCAAGA AAGGATTTAA TGAGAATGAA 540 AAGTTTGCAG CCTTTGACTT CTTCCTTGGA GTCACAGATG AAAGCGCGCT TGAGCCTAAG 600 GGTGGAGTCA AGAGAGCTCC AACAAAAGCA G 631

The polypeptide has a deduced amino acid sequence corresponding to SEQ. ID. No. 22 as follows: Met Ala Ser Gln Val Gly Lys Leu Pro Gly Glu Ser 1               5                   10 Asn Glu Ala Tyr Glu Ala Arg Leu Lys Ala Leu Glu         15                  20 Leu Ala Arg Ala Gln Lys Ala Pro Glu Val Ser Asn 25                  30                  35 Gln Pro Pro Thr Leu Gly Gly Ile Leu Ala Lys Arg             40                  45 Lys Arg Val Ile Glu Asn Ala Leu Ser Lys Thr Val     50                  55                  60 Asp Met Arg Glu Val Leu Arg His Glu Ser Val Val                 65                  70 Leu Ser Pro Asn Val Met Asp Glu Gly Ala Ile Asp         75                  80 Glu Leu Ile Arg Ala Phe Gly Glu Ser Gly Ile Ala 85                  90                  95 Glu Asn Val Gln Phe Asp Val Ala Ile Asp Ile Ala             100                 105 Arg His Cys Ser Asp Val Gly Ser Ser Gln Arg Ser     110                 115                 120 Thr Leu Ile Gly Lys Ser Pro Phe Cys Glu Leu Asn                 125                 130 Arg Ser Glu Ile Ala Gly Ile Ile Arg Glu Val Thr         135                 140 Thr Leu Arg Arg Phe Cys Met Tyr Tyr Ala Lys Ile 145                 150                 155 Val Trp Asn Ile His Leu Glu Thr Gly Ile Pro Pro             160                 165 Ala Asn Trp Ala Lys Lys Gly Phe Asn Glu Asn Glu     170                 175                 180 Lys Phe Ala Ala Phe Asp Phe Phe Leu Gly Val Thr                 185                 190 Asp Glu Ser Ala Leu Glu Pro Lys Gly Gly Val Lys         195                 200 Arg Ala Pro Thr Lys Ala 205                 210

The DNA molecule which constitutes a substantial portion of the RSPaV strain RSP158 genome comprises the nucleotide sequence corresponding to SEQ. ID. No. 23 as follows: GAAGCTAGCA CATTTCTGTT CAACACTATG GCTAACATGT TGTTCACTTT TCTGAGATAT   60 GAACTGACGG GTTCAGAGTC AATAGCATTT GCAGGGGATG ATATGTGTGC TAATAGAAGG  120 TTGCGGCTTA AAACGGAGCA TGAGGGTTTT CTGAACATGA TCTGCCTTAA GGCCAAGGTT  180 CAGTTTGTTT CCAACCCCAC ATTCTGTGGA TGGTGCTTAT TTAAGGAGGG AATCTTCAAG  240 AAACCTCAAC TAATTTGGGA GCGAATATGC ATAGCCAGAG AGATGGGCAA TCTGGAGAAC  300 TGTATTGACA ATTATGCGAT AGAAGTGTCC TATGCATATA GATTGGGTGA GCTATCAATT  360 GAAATGATGA CAGAAGAAGA AGTGGAGGCA CACTACAATT GTGTGAGGTT CCTGGTTAGG  420 AACAAGCATA AGATGAGGTG CTCAATTTCA GGCCTGTTTG AAGTGGTTGA TTAGGCCTTA  480 AGTATTTGGC GTTGTTCGAG TTATTATGAA TAATTTAGTT AAAGCATTAT CAGCCTTCGA  540 GTTTATAGGT GTTTTCAATG TGCTCAAATT TCCAGTTGTT ATACATAGTG TGCCTGGTAG  600 TGGTAAGAGT AGCTTAATAA GGGAATTAAT CTCAGAGGAC GAGAGTTTCG TGGCTTTCAC  660 AGCAGGTGTT CCAGACAGTC CTAACCTCAC AGGGAGGTAC ATCAAGCCTT ACTCACCAGG  720 ATGCGCAGTG CAAGGAAAAG TGAATATACT TGATGAGTAC TTGTCCGTTC AAGACATTTC  780 GGGTTTTGAT GTACTGTTTT CAGACCCGTA CCAGAATATC AGTATTCCCC AAGAGGCGCA  840 TTTCATTAAG TCCAAGACTT GTAGGTTTGG TGTGAACACT TGCAAATACC TTTCCTCTTT  900 CGGTTTCGAA GTTAGCAGCG ACGGGCTGGA CGACGTCATT GTGGGATCGC CCTTCACTCT  960 AGATGTTGAA GGGGTGCTGA TATGTTTTGG CAAGGAGGCG GTAGATCTCG CTGTTGCGCA 1020 CAACTCTGAA TTCAAGTTGC CGTGTGAGGT TCGAGGTTCA ACCTTCAATG TGGTAACCCT 1080 TTTGAAATCA AGAGACCCAA CCCCAGAGGA CAGGCACTGG TTTTACATCG CTGCCACAAG 1140 ACATAGGAAG AAATTGGTCA TTATGCAGTA AAATGCCTTT TCAGCAGCCT GCTAATTGGG 1200 CAAAAACCAT AACTCCATTG ACTATTGGCT TAGGAATTGG ACTTGTGCTG CATTTTCTGA 1260 GAAAGTCAAA TCTACCATAT TCAGGAGACA ACATCCATCA ATTTCCTCAC GGGGGGCGTT 1320 ACCGGGACGG CACAAAAAGT ATAACTTACT GTGGCCCTAA GCAGTCCTTC CCCAGTTCAG 1380 GAATATTTGG TCAGTCTGAG AATTTTGTGC CCTTAATGCT TGTCATAGGT CTAATTGCAT 1440 TCATACATGT ATTGTCTGTT TGGAATTCTG GTCTTGGTAG GAATTGCAAT TGCCATCCAA 1500 ATCCTTGCTC ATGTAGACAA CAGTAGTGGC AGTCACCAAG GTTGCTTTAT CAGGGCCACT 1560 GGAGAGTCTA TTTTGATTGA AAATTGTGGC CCAAGCGAGG CCCTTGCATC AACAGTGAGG 1620 GAGGTGTTGG GGGGTTTGAA GGCTTTAGGA ATTAGCCATA CTACTGAAGA AATTGATTAT 1680 CGTTGTTAAA TTGGTTAAAT GGCGAGTCAA GTTGGTAAGC TCCCCGGAGA ATCAAATGAG 1740 GCATTTGAAG CCCGGCTGAA ATCACTGGAG TTGGCTAGAG CTCAAAAGCA GCCAGAAGGT 1800 TCAAACACAC CGCCTACTCT CAGTGGTGTG CTTGCCAAAC GTAAGAGGGT TATTGAGAAT 1860 GCACTCTCAA AGACAGTGGA CATGAGGGAG GTGTTGAAAC ACGAAACGGT TGTAATTTCC 1920 CCAAATGTCA TGGATGAGGG TGCAATAGAT GAACTGATTC GTGCATTCGG AGAATCAGGC 1980 ATAGCTGAGA GCGCACAATT TGATGTGGC 2009 The RSP158 strain contains five open reading frames (i.e., ORF1-5). ORF1 and ORF5 are only partially sequenced. The nucleotide sequence of RSP158 is 87.6% identical to the corresponding region of RSPaV-1 (type strain). The numbers of amino acid residues of corresponding ORFs of RSP158 and RSPaV-1 (type strain) are exactly the same. In addition, the amino acid sequences of these ORFs have high identities to those of RSPaV-1: 99.3% for ORF1, 95% for ORF2, 99.1% for ORF3, 88.8% for ORF4, and 95.1% for ORF5. The nucleotide and amino acid sequence information of the RSP158 ORFs are described below.

Another DNA molecule of the present invention (RSP158 incomplete ORF1) includes nucleotides 1-447 of SEQ. ID. No. 23. This DNA molecule is believed to code for a polypeptide portion of a RSP158 replicase and comprises a nucleotide sequence corresponding to SEQ. ID. No. 24 as follows: GAAGCTAGCA CATTTCTGTT CAACACTATG GCTAACATGT TGTTCACTTT TCTGAGATAT  60 GAACTGACGG GTTCAGAGTC AATAGCATTT GCAGGGGATG ATATGTGTGC TAATAGAAGG 120 TTGCGGCTTA AAACGGAGCA TGAGGGTTTT CTGAACATGA TCTGCCTTAA GGCCAAGGTT 180 CAGTTTGTTT CCAACCCCAC ATTCTGTGGA TGGTGCTTAT TTAAGGAGGG AATCTTCAAG 240 AAACCTCAAC TAATTTGGGA GCGAATATGC ATAGCCAGAG AGATGGGCAA TCTGGAGAAC 300 TGTATTGACA ATTATGCGAT AGAAGTGTCC TATGCATATA GATTGGGTGA GCTATCAATT 360 GAAATGATGA CAGAAGAAGA AGTGGAGGCA CACTACAATT GTGTGAGGTT CCTGGTTAGG 420 AACAAGCATA AGATGAGGTG CTCAATT 447

The polypeptide encoded by the nucleotide sequence of SEQ. ID. No. 24 has a deduced amino acid sequence corresponding to SEQ. ID. No. 25 as follows: Glu Ala Ser Thr Phe Leu Phe Asn Thr Met Ala Asn 1               5                   10 Met Leu Phe Thr Phe Leu Arg Tyr Glu Leu Thr Gly         15                  20 Ser Glu Ser Ile Ala Phe Ala Gly Asp Asp Met Cys 25                  30                  35 Ala Asn Arg Arg Leu Arg Leu Lys Thr Glu His Glu             40                  45 Gly Phe Leu Asn Met Ile Cys Leu Lys Ala Lys Val     50                  55                  60 Gln Phe Val Ser Asn Pro Thr Phe Cys Gly Trp Cys                 65                  70 Leu Phe Lys Glu Gly Ile Phe Lys Lys Pro Gln Leu         75                  80 Ile Trp Glu Arg Ile Cys Ile Ala Arg Glu Met Gly 85                  90                  95 Asn Leu Glu Asn Cys Ile Asp Asn Tyr Ala Ile Glu             100                 105 Val Ser Tyr Ala Tyr Arg Leu Gly Glu Leu Ser Ile     110                 115                 120 Glu Met Met Thr Glu Glu Glu Val Glu Ala His Tyr                 125                 130 Asn Cys Val Arg Phe Leu Val Arg Asn Lys His Lys         135                 140 Met Arg Cys Ser Ile 145

Another DNA molecule of the present invention (RSP158 ORF2) includes nucleotides 506-1171 of SEQ. ID. No. 23. This DNA molecule codes for a first protein or polypeptide of the RSP158 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 26 as follows: ATGAATAATT TAGTTAAAGC ATTATCAGCC TTCGAGTTTA TAGGTGTTTT CAATGTGCTC  60 AAATTTCCAG TTGTTATACA TAGTGTGCCT GGTAGTGGTA AGAGTAGCTT AATAAGGGAA 120 TTAATCTCAG AGGACGAGAG TTTCGTGGCT TTCACAGCAG GTGTTCCAGA CAGTCCTAAC 180 CTCACAGGGA GGTACATCAA GCCTTACTCA CCAGGATGCG CAGTGCAAGG AAAAGTGAAT 240 ATACTTGATG AGTACTTGTC CGTTCAAGAC ATTTCGGGTT TTGATGTACT GTTTTCAGAC 300 CCGTACCAGA ATATCAGTAT TCCCCAAGAG GCGCATTTCA TTAAGTCCAA GACTTGTAGG 360 TTTGGTGTGA ACACTTGCAA ATACCTTTCC TCTTTCGGTT TCGAAGTTAG CAGCGACGGG 420 CTGGACGACG TCATTGTGGG ATCGCCCTTC ACTCTAGATG TTGAAGGGGT GCTGATATGT 480 TTTGGCAAGG AGGCGGTAGA TCTCGCTGTT GCGCACAACT CTGAATTCAA GTTGCCGTGT 540 GAGGTTCGAG GTTCAACCTT CAATGTGGTA ACCCTTTTGA AATCAAGAGA CCCAACCCCA 600 GAGGACAGGC ACTGGTTTTA CATCGCTGCC ACAAGACATA GGAAGAAATT GGTCATTATG 660 CAGTAA 666

The first protein or polypeptide of the RSP158 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 27 as follows: Met Asn Asn Leu Val Lys Ala Leu Ser Ala Phe Glu 1               5                   10 Phe Ile Gly Val Phe Asn Val Leu Lys Phe Pro Val         15                  20 Val Ile His Ser Val Pro Gly Ser Gly Lys Ser Ser 25                  30                  35 Leu Ile Arg Glu Leu Ile Ser Glu Asp Glu Ser Phe             40                  45 Val Ala Phe Thr Ala Gly Val Pro Asp Ser Pro Asn     50                  55                  60 Leu Thr Gly Arg Tyr Ile Lys Pro Tyr Ser Pro Gly                 65                  70 Cys Ala Val Gln Gly Lys Val Asn Ile Leu Asp Glu         75                  80 Tyr Leu Ser Val Gln Asp Ile Ser Gly Phe Asp Val 85                  90                  95 Leu Phe Ser Asp Pro Tyr Gln Asn Ile Ser Ile Pro             100                 105 Gln Glu Ala His Phe Ile Lys Ser Lys Thr Cys Arg     110                 115                 120 Phe Gly Val Asn Thr Cys Lys Tyr Leu Ser Ser Phe                 125                 130 Gly Phe Glu Val Ser Ser Asp Gly Leu Asp Asp Val         135                 140 Ile Val Gly Ser Pro Phe Thr Leu Asp Val Glu Gly 145                 150                 155 Val Leu Ile Cys Phe Gly Lys Glu Ala Val Asp Leu             160                 165 Ala Val Ala His Asn Ser Glu Phe Lys Leu Pro Cys     170                 175                 180 Glu Val Arg Gly Ser Thr Phe Asn Val Val Thr Leu                 185                 190 Leu Lys Ser Arg Asp Pro Thr Pro Glu Asp Arg His         195                 200 Trp Phe Tyr Ile Ala Ala Thr Arg His Arg Lys Lys 205                 210                 215 Leu Val Ile Met Gln             220 The first protein or polypeptide of the RSP158 triple gene block has a molecular weight of about 20 to 26 kDa., preferably 24.4 kDa.

Another DNA molecule of the present invention (RSP158 ORF3) includes nucleotides 1173-1526 of SEQ. ID. No. 23. This DNA molecule codes for a second protein or polypeptide of the RSP158 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 28 as follows: ATGCCTTTTC AGCAGCCTGC TAATTGGGCA AAAACCATAA CTCCATTGAC TATTGGCTTA  60 GGAATTGGAC TTGTGCTGCA TTTTCTGAGA AAGTCAAATC TACCATATTC AGGAGACAAC 120 ATCCATCAAT TTCCTCACGG GGGGCGTTAC CGGGACGGCA CAAAAAGTAT AACTTACTGT 180 GGCCCTAAGC AGTCCTTCCC CAGTTCAGGA ATATTTGGTC AGTCTGAGAA TTTTGTGCCC 240 TTAATGCTTG TCATAGGTCT AATTGCATTC ATACATGTAT TGTCTGTTTG GAATTCTGGT 300 CTTGGTAGGA ATTGCAATTG CCATCCAAAT CCTTGCTCAT GTAGACAACA GTAG 354

The second protein or polypeptide of the RSP158 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 29 as follows: Met Pro Phe Gln Gln Pro Ala Asn Trp Ala Lys Thr 1               5                   10 Ile Thr Pro Leu Thr Ile Gly Leu Gly Ile Gly Leu         15                  20 Val Leu His Phe Leu Arg Lys Ser Asn Leu Pro Tyr 25                  30                  35 Ser Gly Asp Asn Ile His Gln Phe Pro His Gly Gly             40                  45 Arg Tyr Arg Asp Gly Thr Lys Ile Thr Tyr Cys Gly     50                  55                  60 Pro Lys Gln Ser Phe Pro Ser Ser Gly Ile Phe Gly                 65                  70 Gln Ser Glu Asn Phe Val Pro Leu Met Leu Val Ile         75                  80 Gly Leu Ile Ala Phe Ile His Val Leu Ser Val Trp 85                  90                  95 Asn Ser Gly Leu Gly Arg Asn Cys Asn Cys His Pro             100                 105 Asn Pro Cys Ser Cys Arg Gln Gln     110                 115 The second protein or polypeptide of the RSP158 triple gene block has a molecular weight of about 10 to 15 kDa., preferably 12.9 kDa.

Another DNA molecule of the present invention (RSP158 ORF4) includes nucleotides 1447-1689 of SEQ. ID. No. 23. This DNA molecule codes for a third protein or polypeptide of the RSP158 triple gene block and comprises a nucleotide sequence corresponding to SEQ. ID. No. 30 as follows: ATGTATTGTC TGTTTGGAAT TCTGGTCTTG GTAGGAATTG CAATTGCCAT CCAAATCCTT  60 GCTCATGTAG ACAACAGTAG TGGCAGTCAC CAAGGTTGCT TTATCAGGGC CACTGGAGAG 120 TCTATTTTGA TTGAAAATTG TGGCCCAAGC GAGGCCCTTG CATCAACAGT GAGGGAGGTG 180 TTGGGGGGTT TGAAGGCTTT AGGAATTAGC CATACTACTG AAGAAATTGA TTATCGTTGT 240 TAA 243

The third protein or polypeptide of the RSP158 triple gene block has a deduced amino acid sequence corresponding to SEQ. ID. No. 31 as follows: Met Tyr Cys Leu Phe Gly Ile Leu Val Leu Val Gly 1               5                   10 Ile Ala Ile Ala Ile Gln Ile Leu Ala His Val Asp         15                  20 Asn Ser Ser Gly Ser His Gln Gly Cys Phe Ile Arg 25                  30                  35 Ala Thr Gly Glu Ser Ile Leu Ile Glu Asn Cys Gly             40                  45 Pro Ser Glu Ala Leu Ala Ser Thr Val Arg Glu Val     50                  55                  60 Leu Gly Gly Leu Lys Ala Leu Gly Ile Ser His Thr                 65                  70 Thr Glu Glu Ile Asp Tyr Arg Cys         75                  80 The third protein or polypeptide of the RSP158 triple gene block has a molecular weight of about 5 to 10 kDa., preferably 8.4 kDa.

Yet another DNA molecule of the present invention (RSP158 ORF5) includes nucleotides 1699-2009 of SEQ. ID. No. 23. This DNA molecule codes for a partial RSP158 coat protein or polypeptide and comprises a nucleotide sequence corresponding to SEQ. ID. No. 32 as follows: ATGGCGAGTC AAGTTGGTAA GCTCCCCGGA GAATCAAATG AGGCATTTGA AGCCCGGCTG 60 AAATCACTGG AGTTGGCTAG AGCTCAAAAG CAGCCAGAAG GTTCAAACAC ACCGCCTACT 120 CTCAGTGGTG TGCTTGCCAA ACGTAAGAGG GTTATTGAGA ATGCACTCTC AAAGACAGTG 180 GACATGAGGG AGGTGTTGAA ACACGAAACG GTTGTAATTT CCCCAAATGT CATGGATGAG 240 GGTGCAATAG ATGAACTGAT TCGTGCATTC GGAGAATCAG GCATAGCTGA GAGCGCACAA 300 TTTGATGTGG C 311

The polypeptide has a deduced amino acid sequence corresponding to SEQ. ID. No. 33 as follows: Met Ala Ser Gln Val Gly Lys Leu Pro Gly Glu Ser Asn Glu Ala Phe 1               5                   10                  15 Glu Ala Arg Leu Lys Ser Leu Glu Leu Ala Arg Ala Gln Lys Gln Pro             20                  25                  30 Glu Gly Ser Asn Thr Pro Pro Thr Leu Ser Gly Val Leu Ala Lys Arg         35                  40                  45 Lys Arg Val Ile Glu Asn Ala Leu Ser Lys Thr Val Asp Met Arg Glu     50                  55                  60 Val Leu Lys His Glu Thr Val Val Ile Ser Pro Asn Val Met Asp Glu 65                  70                  75                  80 Gly Ala Ile Asp Glu Leu Ile Arg Ala Phe Gly Glu Ser Gly Ile Ala                 85                  90                  95 Glu Ser Ala Gln Phe Asp Val             100

The following seven cDNA clones are located at the central part of the ORF1 of RSPaV-1 and all have high identities (83.6-98.4%) in nucleotide sequence with the comparable regions of RSPaV-1. When their nucleotide sequences are aligned with MegAlign (DNAStar), a highly conserved region of ca. 600 nucleotides was found. The universal primers BM98-3F/BM98-3R (SEQ. ID. Nos. 51 and 52, infra) were designed based on the conserved nucleotide sequences of this region.

Portions of the genome from yet other strains of Rupestris stem pitting associated viruses have also been isolated and sequenced. These include strains designated 140/94-19 (T7+R1), 140/94-24 (T7+R1), 140/94-2 (T3+F1), 140/94+42 (T7+R1), 140/94-64 (T+R1), 140-94-72 (T7+R1), and 140/94-6 (T3+BM98-3F+F2).

The nucleotide sequence of 140/94-19 (T7+R1) corresponds to SEQ. ID. No. 34 as follows: GCAGGATTGA AGGCTGGCCA CTGTGTGATT TTTGATGAGG TCCAGTTGTT TCCTCCTGGA 60 TACATCGATC TATGCTTGCT TATTATACGT AGTGATGCTT TCATTTCACT TGCCGGTGAT 120 CCATGTCAAA GCACATATGA TTCGCAAAAG GATCGGGCAA TTTTGGGCGC TGAGCAGAGT 180 GACATACTTA GAATGCTTGA GGGCAAAACG TATAGGTATA ACATAGAAAG CAGGAGGTTT 240 GTGAACCCAA TGTTCGAATC AAGACTGCCA TGTCACTTCA AAAAGGGTTC GATGACTGCC 300 GCTTTCGCTG ATTATGCAAT CTTCCATAAT ATGCATGACT TTCTCCTGGC GAGGTCAAAA 360 GGTCCTTTGG ATGCCGTTTT GGTTTCCAGT TTTGAGGAGA AAAAGATAGT CCAGTCCTAC 420 TTTGGAATGA AACAGCTCAC ACTCACATTT GGTGAATCAA CTGGGTTGAA TTTCAAAAAT 480 GGGGGAATTC TCATATCACA TGATTCCTTT CAGACAGATG ATCGGCCGGT GGCTTACTGC 540 TTTATCTCGC TTCAGCCACA ATTTGGATTT GGTGAACATT ACAGGTCTGA GGGTGGAAAG 600 TTTCCTCTCG CACTTTGCTG GCAAACCCCT CTACCATTTT TTAACAGCCA AAAGTGGGGA 660 GAATGTCATA GGAGATTTGC TCCCAGGTGA GCCTAACTTC TTCAGTGGCT TTAACGTTAG 720 CATTGGAAAG AATGAAGGTG TTAGGGAGGA GAAGTTATGT GGTGACCCAT GGTTAAAAGT 780 CATGCTTTTC CTGGGTCAAG ATGAGGATTG TGAAGTTGAA GAGATGGAGT CAGAGTGCTC 840 AAATGAAGAA TGGTTTAAAA CCCACATTCC CCTGAGTAAT CTGGAGTCAA CCAGGGCTAG 900 GTGGGTGGGT AAAATGGCTT TGAAAGAGTA TCGGGAGGTG CGTTGTGGTT ATGAAATGAC 960 TCAACAATTC TTTGATGAGC ATAGGGGTGG AACTGGTGAG CAACTGAGCA ATGCATGTGA 1020 GAGGTTTGAA AGCATTTACC CAAGGCATAA AGGAAATGAT TCAATAACCT TCCTTATGGC 1080 TGTCCGAAAG CGTCTCAAAT TTTCGAAGCC CCAGGTTGAA GCTGCCAAAC TGAGGCGGGC 1140 CAAACCATAT GGGAAATTCT TATTAGACTT TCCTATCCAA AATCCCATTG AAAGCCAGTC 1200 ATAATT 1206

The nucleotide sequence of 140/94-24 (T7+R1) corresponds to SEQ. ID. No. 35 as follows: ATTAACCCAA ATGGTAAGAT TTCCGCCTTG TTTGATATAA CCAATGAGCA CATAAGGCAT 60 GTTGAGAAGA TCGGCAATGG CCCTCAGAGC ATAAAAGTAG ATGAGTTGAG GAAGGTTAAG 120 CGATCCGCCC TTGATCTTCT TTCAATGAAT GGGTCCAAAA TAACCTATTT TCCAAACTTT 180 GAGCGGGCTG AAAAGTTGCA AGGGTGCTTG CTAGGGGGCC TAACTGGTGT CATAAGTGAT 240 GAAAAGTTCA GTGATGCAAA ACCCTGGCTT TCTGGTATAT CAACTGCGGA TATAAAGCCA 300 AGAGAGCTAA CTGTCGTGCT TGGCACTTTT GGGGCTGGAA AGAGTTTCTT GTATAAGAGT 360 TTCATGAAGA GATCTGAGGG AAAATTTGTA ACTTTTGTTT CCCCTAGACG AGCCTTGGCA 420 AATTCAATCA AAAATGATCT TGAAATGGAT GATGGCTGCA AAGTTGCCAA AGCAGGCAAA 480 TCAAAGAAGG AAGGGTGGGA TGTAGTGACC TTTGAAGTTT TCCTTAGAAA AGTTTCTGGT 540 TTGAAAGCTG GTCATTGTGT GATTTTTGAT GAGGTTCAGT TGTTTCCCCC TGGATACATC 600 GATCTGTGTT TACTTGTCAT ACGAAGTGAT GCTTTCATTT CACTTGCTGG TGATCCATGC 660 CAGAGGACAT ATGATTCACA GAAGGATCGA GCAATTTTGG GAGCTGAGCA GAGTGACATA 720 CTCAGACTGC TTGAAGGAAA GACATATAGG TACAACATAG AAAGCAGACG TTTTGTGAAC 780 CCAATGTTTG AATCTAGACT ACCATGTCAC TTCAAAAAGG GTTCAATGAC TGCAGCCTTT 840 GCTGATTATG CAATCTTCCA CAATATGCAT GACTTCCTCC TGGCGAGGTC AAAAGGCCCC 900 TTGGATGCTG TTCTAGTTTC CAGTTTTGAG GAGAAGAAAA TAGTCCAATC CTACTTTGGG 960 ATGAAGCAAC TCACTCTCAC ATTTGGTGAA TCAACTGGGT TGAACTTCAA AAATGGAGGA 1020 ATTCTCATAT CACATGACTC CTTTCATACT GACGATCGAC GGTGGCTTAC TGCTTTATCT 1080 CGATTCAGCC ATAATTTGGA TTTGGTGAAC ATCACAGGTC TTGAGGGTGG AAAGTTTTCT 1140 CTCACATTTT GCTGGTAAAC CCCTTTACCA CTTTTTGACG GCTTAAAAGT GGAGAGAATG 1200 TCATACGAGA CCTGCTTCAG GTGAGCCTAA CTTCTTTTAG GGGTTCAATG TCAGCATTGG 1260 AAAAAAATGG AAGGGGTTAG AGAA 1284

The nucleotide sequence of 140/94-2 (T3+F1) corresponds to SEQ. ID. No. 36 as follows: CATTTTTAAA ATTTAATCCA GTCGACTCAC CAAATGTGAG CGTAAGCTGT TTCATCCCAA 60 AGTAGGACTG GACTATTTTC TTCTCCTCAA AACTAGAAAC CAGAATGGCA TCCAAAGGAC 120 CTTTTGACCT TGCCAGGAGG AAATCATGCA TATTGTGGAA AATGGCATAA TCAGCAAAGG 180 CAGCAGTCAT TGTACCCTTT TTGAAGTGAC ATGGCAGTCG AGATTCAAAC ATTGGGTTCA 240 CAAATCTTCT GCTTTCTATG TTGTACCTAT ACGTCTTGCC TTCAAGTATT TTGAGTATGT 300 CACTCTGCTC AGCGCCCAAA ATCGCCCGAT CTTTTTGTGA GTCATATGTG CTCTGACATG 360 GGTCACCAGC AAGTGAAATG AAAGCATCAC TACGTATAAT AAGCAAACAT AGATCGATGT 420 ATCCAGGGGG AAACAACTGG ACCTCATCGA AAATTACACA GTGACCAGCT TTTAGACCTG 480 CAACTTTTCT AAGGAAGACT TCAAAAGTCA CAACATCCCA TCCTTCCTTC TTTGACCTGC 540 CTGCTTTGGC AACTTTGCAG CTATCATCCA TTTCAAGATC ATTTTTGATT GAATTCGCTA 600 GAGCCCGTCT GGGGGAAACA AAAGTTACGA ATTTACCCTC AGATCTTTTC ATAAAGCTCT 660 TGTACAAAAA GCTTTTTCCG GCTCCAAATG TGCCAAGCAC AACAGTTAGC TCCCTCGGCT 720 TAATGTCAGT AGTTGATATA CCAGAAAGCC AGGGCTTTGC ATCACTGAAC TTCTCATCAC 780 TTATGACACC AGTTAGGCCT CCTAGCAGAC ACCCTTGCAA CTTTTCAGCC CGCTCAAAAC 840 TTGGGAAGTA GGTTACCTTG GACCCATTAA TTGAAAGAAG ATCAAGGGCG GATCGCTTGA 900 CCTTTCGCAA TTCATCTACT TTAATGCTCT GAGGGCCATT ACCTATCTTT TCAACATGCC 960 TTATGTGCTC ATTAGTTATG TCAAACAGAG CGGAAAACTT GCCATGTGGA TTAATCACCT 1020 CAATTTCCCC ATTTATGTCA CACTTAGCGC AAATGTCAAA AGCCTCAAAG GCTTCAGCTA 1080 AGTTACATCA TGTTGAGCCT CCCCCTTGGC AAAGCTCCTC AAAAATGTGG TTAGTGCTAG 1140 GCCTGCACAA TAATTAACAC ATCAACTTCA CCCTGCCAAT GCTGAACAAT ACTGTTATCA 1200 TGCAACCATC CATGGGGCAC ATGGTTGGAA TTGATTGATT TAAGGCAAAA ATCCCCACAG 1260 GGGGCATCCC CTTCCCCAAT TTCCACTGAT TCATACTCTG GCGTTATCAT ATCAACCCAA 1320 TGTGTCAAAT ACAAATAATG CAATCTCTCA TCTCCGATAA CATTTCCCCC ATTTTTTAAA 1380 ATGGTGGGG TGAAAATTGG AA 1402

The nucleotide sequence of 140/94-42 (T7+R1) corresponds to SEQ. ID. No. 37 as follows: GTGGTTTTTG CAACAACAGG CCCAGGTCTA TCTAAGGTTT TGGAAATGCC TCGAAGCAAG 60 AAGCAATCTA TTCTGGTTCT TGAGGGAGCC CTATCCATAG AAACGGACTA TGGCCCAAAA 120 GTTCTGGGAT CTTTTGAAGT TTTCAAAGGG GATTTCAACA TTAAAAAAAT GGAAGAAAGT 180 TCCATCTTTG TAATAACATA CAAGGCCCCA GTTAGATCTA CTGGCAAGTT GAGGGTCCAC 240 CAATCAGAAT GCTCATTTTC TGGATCCAAG GAGGTATTGC TGGGTTGTCA GATTGAGGCA 300 TGTGCTGATT ATGATATTGA TGATTTCAAT ACTTTCTTTG TACCTGGTGA TGGTAATTGC 360 TTTTGGCATT CAGTTGGTTT CTTACTCAGT ACTGACGGAC TTGCTTTGAA GGCCGGCATT 420 CGTTCTTTCG TGGAGAGTGA ACGCCTGGTG AGTCCAGATC TTTCAGCCCC AACCATTTCT 480 AAACAACTGG GGGAAAATGC TTATGCCGAG AATGAGATGA TTGCATTATT TTGTATTCGA 540 CACCATGTGA GGCTGATAGT GATTACGCCA GAGTATGAAG TCAGTTGGAA ATTTGGGGAA 600 GGTGAATGGC CCCTGTGCGG AATTCTTTGC CTTAAATCAA ATCACTTCCA ACCATGTGCC 660 CCATTGAATG GTTGCATGAT TACAGCTATT GCTTCAGCAC TTGGTAGGCG TGAAGTTGAT 720 GTGCTTAATT ATCTGTGCAG GCCTAGCACT AACCACATTT TTGAGGAGCT TTGCCAAGGG 780 GGAGGCCTCA ACATGATGTA CTTAGCTGAA GCCTTTGAGG CTTTTGACAT TTGCGCTAAG 840 TGTGACATAA ATGGGGAAAT TGAGGTGATT AATCCACATG GCAAGTTTTC CGCTCTGTTT 900 GACATAACTA ATGAGCACAT AAGGCATGTT GAAAAGATAG GTAATGGCCC TCAGAGCATT 960 AAAGTAGATG AATTGCGAAA GGTCAAGCGA TCTGCCCTTG ATCTTCTTTC AATTAATGGG 1020 TCCAAGGTAA CCTACTTCCC AAGTTTTGAG CGGGCTGAAA AGTTGCAAGG GTGTCTGCTA 1080 GGAGGCCTAA CTGGTGTCAT AAGTGATGAG AAAGTCAGTG ATGCAAAGCC CTGCTTTTTG 1140 GTATATCAAC TACTGACATT AAGCCGAGGG AGCTAACTGT TGTGCTTTGG CACATTTGGA 1200 GCCCGGAAAA AGCCTTTTGT ACCAAGAGCT TTATTG 1236

The nucleotide sequence of 140/94-6 (T3+BM98-3F+F2) corresponds to SEQ. ID. No. 38 as follows: GTCTAACTGG CGTTATAAGT GATGAGAAAT TCAGTGATGC AAAACCTTGG CTTTCTGGTA 60 TATCTACTAC AGATATTAAG CCAAGGGAAT TAACTGTTGT GCTTGGTACA TTTGGGGCTG 120 GGAAGAGTTT CTTGTACAAG AGTTTGATGA AAAGGTCTGA GGGTAAATTC GTAACCTTTG 180 TTTCTCCCAG ACGTGCTTTA GCAAATTCAA TCAAAAATGA TCTTGAAATG GATGATAGCT 240 GCAAAGTTGC CAAAGCAGGT AGGTCAAAGA AGGAAGGGTG GGATGTAGTA ACTTTTGAGG 300 TCTTCCTCAG AAAAGTTGCA GGATTGAAGG CTGGCCACTG TGTGATTTTT GATGAGGTCC 360 AGTTGTTTCC TCCTGGATAC ATCGATCTAT GCTTGCTTAT TATACGTAGT GATGCTTTCA 420 TTTCACTTGC CGGTGATCCA TGTCAAAGGA CATATGATTC GCAAAAGGAT CGGGCAATTT 480 TGGGCGCTGA GCAGAGTGAC ATACTTAGAA TGCTTGAGGG CAAAACGTAT AGGTATAACA 540 TAGAAAGCAG GAGGTTTGTG AACCCAATGT TCGAATCAAG ACTGCCATGT CACTTCAAAA 600 AGGGTTCGAT GACTGCCGCT TTCGCTGATT ATGCAATCTT CCATAATATG CATGACTTTC 660 TCCTGGCGAG GTCAAAAGGT CCTTTGGATG CCGTTTTGGT TTCCAGTTTT GAGGAGAAAA 720 AGATAGTCCA GTCCTACTTT GGAATGAAAC AGCTCACACT CACATTTGGT GAATCAACTG 780 GGTTGAATTT CAAAAATGGG GGAATTCTCA TATCACATGA TTCCTTTCAC ACAGATGATC 840 GGCGGTGGCT TACTGCTTTA TCTCGCTTCA GCCACAATTT GGATTTGGTG AACATTACAG 900 GTCTGAGGTG GAAAGTTTCC TCTCGCACTT TGCTGGCAAA CCCCTCTACC ATTTTTTAAC 960 AGCCAAAAGT GGGGAGAATG TCATACGAGA TTTGCTCCCA GGTGAGCCTA ACTTCTTCAG 1020 TGGCTTTAAC GTTAGCATTG GAAAGAATGA AGGTGTTAGG GAGGAGAAGT TATGTGGTGA 1080 CCCATGGTTA AAAGTCATGC TTTTCCTGGG TCAAGATGAG GATTGTGAAG TTGAAGAGAT 1140 GGAGTCAGAG TGCTCAAATG AAGAATGGTT TAAAACCCAC ATTCCCCTGA GTAATCTGGA 1200 GTCAACCAGG GCTAGGTGGG TGGGTAAAAT GGCCTTGAAA GAGTATCGGG AGGTGCGTTG 1260 TGGTTATGAA ATGACTCAAC AATTCTTTGA TGACAT 1296

The nucleotide sequence of 140/94-64 (T7+R1) corresponds to SEQ. ID. No. 39 as follows: ATGTTCACCA AATCCAAATT ATGGCTGAAG CGAGATAAAG CAGTAAGCCA CCGCCGATCA 60 TCTGTGTGAA AGGAATCATG TGATATGAGA ATTCCCCCAT TTTTGAAATT CAACCCAGTT 120 GATTCACCAA ATGTGAGTGT GAGCTGTTTC ATTCCAAAGT AGGACTGGAC TATCTTTTTC 180 TCCTCAAAAC TGGAAACCAA AACGGCATCC AAAGGACCTT TTGACCTCGC CAGGAGAAAG 240 TCATGCATAT TATGGAAGAT TGCATAATCA GGGAAAGCGG CAGTCATTGA GCCCTTTTTG 300 AATTGACATG GCAGTCTTGA TTCGAACATT GGATTCACAA ACCTCCTGCT TTCAATGTTA 360 TACCTATACG TCTTGCCCTC AAGCAGTCTA AGTATGTCAC TCTGCTCAGC GCCCAAAATT 420 GCCCGATCCT TTTGCGAATC ATATGTGCTT TGACATGGAT CACCGGCAAG TGAAATGAAA 480 GCATCACTAC GTATAATAAG CAAGCATAGA TCGATGTATC CAGGAGGAAA CAACTGGACC 540 TCATCGAAAA TCACACAGTG GCCAGCCTTC AATCCTGCAA CTTTTCTGAG GAAAACCTCA 600 AAAGTTACTA CATCCCACCC TTCCTTCTTT GACCTACCTG CTTTAGCAAC TTTGCAGCTA 660 TCATCCATTT CAAGATCATT TTTGATTGAA TTTGCTAAAG CACGTCTGGG AGAAACAAAG 720 GTTACGAATT TACCCTCAGA CCTTTTCATG AAACTCTTGT ACAAGAAACT CTTCCCAGCC 780 CCAAATGTAC CAAGCACGAC AGTCAACTCC CTTGGCTTAA TATCAGTAGT AGATATACCA 840 GAAAGCCAAG GTTTTGCATC ACTGAACTTC TCATCACTTA TAACGCCAGT TAGGCCCCCT 900 AGCAAC 907

The nucleotide sequence of 140-94-72 (T7+R1) corresponds to SEQ. ID. No. 40 as follows: AGAATGCTTA TGCTGAGAAT GAGATGATTG CATTATTTTG CATCCGGCAC CATGTAAGGC 60 TTATAGTAAT AACACCGGAA TATGAAGTTA GTTGGAAATT TGGGGAAAGT GAGTGGCCCC 120 TATGTGGAAT TCTTTGCCTG AGGTCCAATC ACTTCCAACC ATGCGCCCCG CTGAATGGTT 180 GCATGATCAC GGCTATTGCT TCAGCACTTG GGAGGCGTGA GGTTGATGTG TTAAATTATC 240 TGTGTAGGCC TAGCACTAAT CACATCTTTG AGGAGCTGTG CCAGGGCGGA GGGCTTAATA 300 TGATGTACTT GGCTGAAGCT TTTGAGGCCT TTGACATTTG TGCAAAGTGC GACATAAATG 360 GGGAAATTGA GGTCATTAAC CCAAATGGCA AGATTTCCGC CTTGTTTGAT ATAACTAATG 420 AGCACATAAG GCATGTTGAG AAGATCAGCA ATGGCCCTCA GAGCATAAAA ATAGATGAGT 480 TGAGGAAGGT TAAGCGATCC CGCCTTGACC TTCTTTCAAT GAATGGGTCC AAAATAACCT 540 ATTTTCCAAA CTTTGAGCGG GCTGAAAAGT TGCAAGGGTG CTTGCTAGAG GGCCTGACTG 600 GTGTCATAAG TGATGAAAAG TTCAGTGATG CAAAACCTTG GCTTTCTGGT ATATCAACTG 660 CGGATATTAA GCCAAGAGAG CTAACTGTCG TGCTTGGCAC ATTTGGTGCT GGAAAGAGTT 720 TCTTGTATAA GAGTTTCATG AAGAGATCTG AAGGAAAATT TGTAACTTTT GTTTCCCCTA 780 GGCGAGCTTT GGCCAATTCG ATCAAGAATG ATCTTGAAAT GGATGATGGC TGCAAAGTTG 840 CCAAAGCAGG CAAGTCAAAG AAGGAAGGGT GGGATGTGGT AACATTTGAG GTTTTCCTTA 900 GAAAAGTTTC TGGTTTGAAG GCTGGTCATT GTGTGATTTT CGATGAGGTT CAGTTGTTTC 960 CCCCTGGATA TATCGATCTA TGTTTACTTG TCATACGCAG TGATGCTTTT ATTTCACTTG 1020 CCGGTGATCC ATGCCAGAGC ACATATGATT CACAAAAGGA TCGGGCAATT TTGGGAGCTG 1080 AGCAGAGTGA CATACTCAGA TTGCTTGAAG GAAAGACGTA TAGGTACAAC ATAGAAAGCA 1140 GACGTTTTGT GAACCCAATG TTTGAATTTA GACTACCATG TCACTTCAAA AAAGGGTTCA 1200 ATGACTGCTG CCTTTGCTGA TTATGCAATC TT

Also encompassed by the present invention are fragments of the DNA molecules of the present invention. Suitable fragments capable of imparting RSP resistance to grape plants are constructed by using appropriate restriction sites, revealed by inspection of the DNA molecule's sequence, to: (i) insert an interposon (Felley et al., “Interposon Mutagenesis of Soil and Water Bacteria: A Family of DNA Fragments Designed for in vitro Insertion Mutagenesis of Gram-negative Bacteria,” Gene 52:147-15 (1987), which is hereby incorporated by reference) such that truncated forms of the RSP virus polypeptide or protein, that lack various amounts of the C-terminus, can be produced or (ii) delete various internal portions of the protein. Alternatively, the sequence can be used to amplify any portion of the coding region, such that it can be cloned into a vector supplying both transcription and translation start signals.

Suitable DNA molecules are those that hybridize to a DNA molecule comprising a nucleotide sequence of at least 15 continuous bases of SEQ. ID. No. 1 under stringent conditions characterized by a hybridization buffer comprising 0.9M sodium citrate (“SSC”) buffer at a temperature of 37° C. and remaining bound when subject to washing with SSC buffer at 37° C.; and preferably in a hybridization buffer comprising 20% formamide in 0.9M saline/0.9M SSC buffer at a temperature of 42° C. and remaining bound when subject to washing at 42° C. with 0.2×SSC buffer at 42° C.

Variants may also (or alternatively) be modified by, for example, the deletion or addition of nucleotides that have minimal influence on the properties, secondary structure and hydropathic nature of the encoded protein or polypeptide. For example, the nucleotides encoding a protein or polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The nucleotide sequence may also be altered so that the encoded protein or polypeptide is conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide.

The protein or polypeptide of the present invention is preferably produced in purified form (preferably, at least about 80%, more preferably 90%, pure) by conventional techniques. Typically, the protein or polypeptide of the present invention is isolated by lysing and sonication. After washing, the lysate pellet is re-suspended in buffer containing Tris-HCl. During dialysis, a precipitate forms from this protein solution. The solution is centrifuged, and the pellet is washed and re-suspended in the buffer containing Tris-HCl. Proteins are resolved by electrophoresis through an SDS 12% polyacrylamide gel.

The DNA molecule encoding the RSP virus protein or polypeptide of the present invention can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e., not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.

U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eukaryotic cells grown in tissue culture.

Recombinant genes may also be introduced into viruses, such as vaccinia virus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.

Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKCO101, SV 40, pBluescript II SK +/− or KS+/− (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see Studier et. al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene Expression Technology, vol. 185 (1990), which is hereby incorporated by reference), and any derivatives thereof. Suitable vectors are continually being developed and identified. Recombinant molecules can be introduced into cells via transformation, transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1982), which is hereby incorporated by reference.

A variety of host-vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria or transformed via particle bombardment (i.e. biolistics). The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.

Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (“mRNA”) translation).

Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eukaryotic promoters differ from those of procaryotic promoters. Furthermore, eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters are not recognized and do not function in eukaryotic cells.

Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eukaryotes; Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which is hereby incorporated by reference.

Promoters vary in their “strength” (i.e., their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P_(R) and P_(L) promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, Ipp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.

Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.

Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promoter, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (“SD”) sequence about 7-9 bases 5′ to the initiation codon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.

Once the isolated DNA molecules encoding the various Rupestris stem pitting associated virus proteins or polypeptides, as described above, have been cloned into an expression system, they are ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.

The present invention also relates to RNA molecules which encode the various RSP virus proteins or polypeptides described above. The transcripts can be synthesized using the host cells of the present invention by any of the conventional techniques. The mRNA can be translated either in vitro or in vivo. Cell-free systems typically include wheat-germ or reticulocyte extracts. In vivo translation can be effected, for example, by microinjection into frog oocytes.

One aspect of the present invention involves using one or more of the above DNA molecules encoding the various proteins or polypeptides of a RSP virus to transform grape plants in order to impart RSP resistance to the plants. The mechanism by which resistance is imparted in not known. In one hypothetical mechanism, the transformed plant can express the coat protein or polypeptide, and, when the transformed plant is inoculated by a RSP virus, such as RSPaV-1, the expressed coat protein or polypeptide surrounds the virus, thereby preventing translation of the viral DNA.

In this aspect of the present invention, the subject DNA molecule incorporated in the plant can be constitutively expressed. Alternatively, expression can be regulated by a promoter which is activated by the presence of RSP virus. Suitable promoters for these purposes include those from genes expressed in response to RSP virus infiltration.

The isolated DNA molecules of the present invention can be utilized to impart RSP virus resistance for a wide variety of grapevine plants. The DNA molecules are particularly well suited to imparting resistance to Vitis scion or rootstock cultivars. Scion cultivars which can be protected include those commonly referred to as Table or Raisin Grapes, such as Alden, Almeria, Anab-E-Shahi, Autumn Black, Beauty Seedless, Black Corinth, Black Damascus, Black Malvoisie, Black Prince, Blackrose, Bronx Seedless, Burgrave, Calmeria, Campbell Early, Canner, Cardinal, Catawba, Christmas, Concord, Dattier, Delight, Diamond, Dizmar, Duchess, Early Muscat, Emerald Seedless, Emperor, Exotic, Ferdinand de Lesseps, Fiesta, Flame seedless, Flame Tokay, Gasconade, Gold, Himrod, Hunisa, Hussiene, Isabella, Italia, July Muscat, Khandahar, Katta, Kourgane, Kishmishi, Loose Perlette, Malaga, Monukka, Muscat of Alexandria, Muscat Flame, Muscat Hamburg, New York Muscat, Niabell, Niagara, Olivette blanche, Ontario, Pierce, Queen, Red Malaga, Ribier, Rish Baba, Romulus, Ruby Seedless, Schuyler, Seneca, Suavis (IP 365), Thompson seedless, and Thomuscat. They also include those used in wine production, such as Aleatico, Alicante Bouschet, Aligote, Alvarelhao, Aramon, Baco blanc (22A), Burger, Cabernet franc, Cabernet, Sauvignon, Calzin, Carignane, Charbono, Chardonnay, Chasselas dore, Chenin blanc, Clairette blanche, Early Burgundy, Emerald Riesling, Feher Szagos, Fernao Pires, Flora, French Colombard, Fresia, Furmint, Gamay, Gewurztraminer, Grand noir, Gray Riesling, Green Hungarian, Green Veltliner, Grenache, Grillo, Helena, Inzolia, Lagrein, Lambrusco de Salamino, Malbec, Malvasia bianca, Mataro, Melon, Merlot, Meunier, Mission, Montua de Pilas, Muscadelle du Bordelais, Muscat blanc, Muscat Ottonel, Muscat Saint-Vallier, Nebbiolo, Nebbiolo fino, Nebbiolo Larnpia, Orange Muscat, Palomino, Pedro Ximenes, Petit Bouschet, Petite Sirah, Peverella, Pinot noir, Pinot Saint-George, Primitivo di Gioa, Red Veltliner, Refosco, Rkatsiteli, Royalty, Rubired, Ruby abernet, Saint-Emilion, Saint Macaire, Salvador, Sangiovese, Sauvignon blanc, Sauvignon gris, Sauvignon vert, Scarlet, Seibel 5279, Seibel 9110, Seibel 13053, Semillon, Servant, Shiraz, Souzao, Sultana Crimson, Sylvaner, Tannat, Teroldico, Tinta Madeira, Tinto cao, Touriga, Traminer, Trebbiano Toscano, Trousseau, Valdepenas, Viognier, Walschriesling, White Riesling, and Zinfandel. Rootstock cultivars which can be protected include Couderc 1202, Couderc 1613, Couderc 1616, Couderc 3309, Dog Ridge, Foex 33 EM, Freedom, Ganzin 1 (A×R #1), Harmony, Kober 5BB, LN33, Millardet & de Grasset 41B, Millardet & de Grasset 420A, Millardet & de Grasset 101-14, Oppenheim 4 (SO₄), Paulsen 775, Paulsen 1045, Paulsen 1103, Richter 99, Richter 110, Riparia Gloire, Ruggeri 225, Saint-George, Salt Creek, Teleki 5A, Vitis rupestris Constantia, Vitis California, and Vitis girdiana.

Plant tissue suitable for transformation include leaf tissue, root tissue, meristems, zygotic and somatic embryos, and anthers. It is particularly preferred to utilize embryos obtained from anther cultures.

The expression system of the present invention can be used to transform virtually any plant tissue under suitable conditions. Tissue cells transformed in accordance with the present invention can be grown in vitro in a suitable medium to impart RSPaV resistance. Transformed cells can be regenerated into whole plants such that the protein or polypeptide imparts resistance to RSPaV in the intact transgenic plants. In either case, the plant cells transformed with the recombinant DNA expression system of the present invention are grown and caused to express that DNA molecule to produce one of the above-described RSPaV proteins or polypeptides and, thus, to impart RSPaV resistance.

In producing transgenic plants, the DNA construct in a vector described above can be microinjected directly into plant cells by use of micropipettes to transfer mechanically the recombinant DNA. Crossway, Mol. Gen. Genetics, 202:179-85 (1985), which is hereby incorporated by reference. The genetic material may also be transferred into the plant cell using polyethylene glycol. Krens, et al., Nature, 296:72-74 (1982), which is hereby incorporated by reference.

One technique of transforming plants with the DNA molecules in accordance with the present invention is by contacting the tissue of such plants with an inoculum of a bacteria transformed with a vector comprising a gene in accordance with the present invention which imparts RSPaV resistance. Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28° C.

Bacteria from the genus Agrobacterium can be utilized to transform plant cells. Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacterium tumefaciens (e.g., strains C58, LBA4404, or EHA105) is particularly useful due to its well-known ability to transform plants.

Heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of A. tumefaciens or the R1 plasmid of A. rhizogenes. The Ti or R1 plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome. J. Schell, Science, 237:1176-83 (1987), which is hereby incorporated by reference.

After transformation, the transformed plant cells must be regenerated.

Plant regeneration from cultured protoplasts is described in Evans et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co., New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. III (1986), which are hereby incorporated by reference.

It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugarcane, sugar beets, cotton, fruit trees, and legumes.

Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.

After the expression cassette is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

Once transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure so that the DNA construct is present in the resulting plants. Alternatively, transgenic seeds are recovered from the transgenic plants. These seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants.

Another approach to transforming plant cells with a gene which imparts resistance to pathogens is particle bombardment (also known as biolistic transformation) of the host cell. This can be accomplished in one of several ways. The first involves propelling inert or biologically active particles at cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., and in Emerschad et al., “Somatic Embryogenesis and Plant Development from Immature Zygotic Embryos of Seedless Grapes (Vitis vinifera),” Plant Cell Reports, 14:6-12 (1995) (“Emerschad (1995)”), which are hereby incorporated by reference. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into plant cells.

Once a grape plant tissue is transformed in accordance with the present invention, it is regenerated to form a transgenic grape plant. Generally, regeneration is accomplished by culturing transformed tissue on medium containing the appropriate growth regulators and nutrients to allow for the initiation of shoot meristems. Appropriate antibiotics are added to the regeneration medium to inhibit the growth of Agrobacterium and to select for the development of transformed cells. Following shoot initiation, shoots are allowed to develop tissue culture and are screened for marker gene activity.

The DNA molecules of the present invention can be made capable of transcription to a messenger RNA that does not translate to the protein. This is known as RNA-mediated resistance. When a Vitis scion or rootstock cultivar is transformed with such a DNA molecule, the DNA molecule can be transcribed under conditions effective to maintain the messenger RNA in the plant cell at low level density readings. Density readings of between 15 and 50 using a Hewlet ScanJet and Image Analysis Program are preferred.

A portion of one or more DNA molecules of the present invention as well as other DNA molecules can be used in a transgenic grape plant in accordance with U.S. patent application Ser. No. 09/025,635, which is hereby incorporated herein by reference.

The RSPaV protein or polypeptide can also be used to raise antibodies or binding portions thereof or probes. The antibodies can be monoclonal or polyclonal.

Monoclonal antibody production may be effected by techniques which are well-known in the art. Basically, the process involves first obtaining immune cells (lymphocytes) from the spleen of a manunal (e.g., mouse) which has been previously immunized with the antigen of interest either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with (mouse) myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody. A description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein, Nature, 256:495 (1975), which is hereby incorporated by reference.

Mammalian lymphocytes are immunized by in vivo immunization of the animal (e.g., a mouse) with the protein or polypeptide of the present invention. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. Following the last antigen boost, the animals are sacrificed and spleen cells removed.

Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol (“PEG”) or other fusing agents. (See Milstein and Kohler, Eur. J. Immunol., 6:511 (1976), which is hereby incorporated by reference.) This immortal cell line, which is preferably murine, but may also be derived from cells of other mammalian species, including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and to have good fusion capability. Many such cell lines are known to those skilled in the art, and others are regularly described.

Procedures for raising polyclonal antibodies are also well known. Typically, such antibodies can be raised by administering the protein or polypeptide of the present invention subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. The antigens can be injected at a total volume of 100 μl per site at six different sites. Each injected material will contain synthetic surfactant adjuvant pluronic polyols, or pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis. The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. Ultimately, the rabbits are euthanized with pentobarbital 150 mg/Kg IV. This and other procedures for raising polyclonal antibodies are disclosed in Harlow et. al., editors, Antibodies: A Laboratory Manual (1988), which is hereby incorporated by reference.

In addition to utilizing whole antibodies, binding portions of such antibodies can be used. Such binding portions include Fab fragments, F(ab′)₂ fragments, and Fv fragments. These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in Goding, Monoclonal Antibodies: Principles and Practice, New York: Academic Press, pp. 98-118 (1983), which is hereby incorporated by reference.

The present invention also relates to probes found either in nature or prepared synthetically by recombinant DNA procedures or other biological procedures. Suitable probes are molecules that bind to RSP viral antigens identified by the polyclonal antibodies of the present invention or bind to the nucleic acid of RSPaV. Such probes can be, for example, proteins, peptides, lectins, or nucleic acids.

The antibodies or binding portions thereof or probes can be administered to RSPaV infected scion cultivars or rootstock cultivars. Alternatively, at least the binding portions of these antibodies can be sequenced, and the encoding DNA synthesized. The encoding DNA molecule can be used to transform plants together with a promoter which causes expression of the encoded antibody when the plant is infected by an RSPaV. In either case, the antibody or binding portion thereof or probe will bind to the virus and help prevent the usual stem pitting response.

Antibodies raised against the proteins or polypeptides of the present invention or binding portions of these antibodies can be utilized in a method for detection of RSPaV in a sample of tissue, such as tissue from a grape scion or rootstock. Antibodies or binding portions thereof suitable for use in the detection method include those raised against a replicase, proteins or polypeptides of the triple gene block, or a coat protein or polypeptide in accordance with the present invention. Any reaction of the sample with the antibody is detected using an assay system which indicates the presence of RSPaV in the sample. A variety of assay systems can be employed, such as enzyme-linked immunosorbent assays, radioimmunoassays, gel diffusion precipitin reaction assays, immunodiffusion assays, agglutination assays, fluorescent immunoassays, protein A immunoassays, or immunoelectrophoresis assays.

Alternatively, the RSPaV can be detected in such a sample using the DNA molecules of the present, RNA molecules of the present invention, or DNA or RNA fragments thereof, as probes in nucleic acid hybridization assays for detecting the presence of complementary virus DNA or RNA in the various tissue samples described above. The nucleotide sequence is provided as a probe in a nucleic acid hybridization assay or a gene amplification detection procedure (e.g., using a polymerase chain reaction procedure). The nucleic acid probes of the present invention may be used in any nucleic acid hybridization assay system known in the art, including, but not limited to, Southern blots (Southern, E. M., “Detection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresis,” J. Mol. Biol. 98:503-17 (1975), which is hereby incorporated by reference), Northern blots (Thomas, P. S., “Hybridization of Denatured RNA and Small DNA Fragrnents Transferred to Nitrocellulose,” Proc. Nat'l Acad. Sci. USA, 77:5201-05 (1980), which is hereby incorporated by reference), and Colony blots (Grunstein, M., et al., “Colony Hybridization: A Method for the Isolation of Cloned cDNAs that Contain a Specific Gene,” Proc. Nat'l Acad. Sci. USA, 72:3961-65 (1975), which is hereby incorporated by reference). Alternatively, the isolated DNA molecules of the present invention or RNA transcripts thereof can be used in a gene amplification detection procedure (e.g., a polymerase chain reaction). Erlich, H. A., et. al., “Recent Advances in the Polymerase Chain Reaction,” Science 252:1643-51 (1991), which is hereby incorporated by reference. Any reaction with the probe is detected so that the presence of RSP virus in the sample is indicated. Such detection is facilitated by providing the DNA molecule of the present invention with a label. Suitable labels include a radioactive compound, a fluorescent compound, a chemiluminescent compound, an enzymatic compound, or other equivalent nucleic acid labels.

Depending upon the desired scope of detection, it is possible to utilize probes having nucleotide sequences that correspond with conserved or variable regions of the ORF or UTR. For example, to distinguish RSPaV from other related viruses (as described herein), it is desirable to use probes which contain nucleotide sequences that correspond to sequences more highly conserved among all RSPaV strains. Also, to distinguish between different RSPaV strains (e.g., RSPaV-1, RSP47-4, RSP158), it is desirable to utilize probes containing nucleotide sequences that correspond to sequences less highly conserved among the RSP virus strains.

Nucleic acid (DNA or RNA) probes of the present invention will hybridize to complementary RSPaV-1 nucleic acid under stringent conditions. Less stringent conditions may also be selected. Generally, stringent conditions are selected to be about 50° C. lower than the thermal melting point (T_(m)) for the specific sequence at a defined ionic strength and pH. The T_(m) is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. The T_(m) is dependent upon the solution conditions and the base composition of the probe, and may be calculated using the following equation: T _(m)=79.8° C.+(18.5×Log[Na+])+ +(58.4° C.×%[G+C]) −(820/#bp in duplex) −(0.5×% formamide) Nonspecific binding may also be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein-containing solutions, addition of heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNase. Generally, suitable stringent conditions for nucleic acid hybridization assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected.

The development of a rapid detection method for RSP is a major breakthrough, because the only detection method now available is through inoculation of St. George grape indicators, which takes two to three years to develop symptoms. A serological or nucleic acid based detection tests developed for RSP will take only 1 to 2 days and it is less expensive. The woody indicator test on St. George costs $250 per sample, while a serological or nucleic acid based test would cost $30-50 per sample. Moreover, the rapid tests will speed up the introduction of grape imports into the US from the current three years to about six months. These applications will be valuable wherever grapes are grown. Since RSP is part of the rugose wood complex, development of rapid detection methods will be invaluable in determining the significance of RSP in the rugose wood complex. This will allow an investigator to determine whether RSP alone can cause the rugose wood complex or if other components are needed. In addition, these rapid detection methods are very useful to evaluate the resistance of transgenic plants to Rupestris stem pitting associated virus.

EXAMPLES

The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.

Example 1 Grapevine Materials for dsRNA Analysis

Samples from 15 accessions that induced pitting on graft-inoculated St. George were collected from the National Grapevine Germplasm Repository of the USDA Plant Genetic Resources Unit (PGRU) at Geneva and used for dsRNA analysis. Positive controls used included Thompson Seedless (RSP105) (Golino, “The Davis Grapevine Virus Collection,” Am. J. Enology Viticulture, 43:200-05 (1992), which is hereby incorporated by reference) from the FPMS, University of California (Davis) and Pinot Noir (SVP1186-09A2), which was kindly provided by Dr. R. Johnson of Center for Plant Health, Agriculture Canada, Sidney, British Columbia. Negative controls as judged by indexing on St. George included Freedom from the PGRU at Geneva, N.Y., and Verduzzo 233A. The latter was kindly provided by Dr. P. Silvano of the Sezione di Fitovirologia, ERSA Servizio Chimico-Agrario e della Certificazione, Pozzuolo del Friuh (UD), Italy.

Example 2 Grapevine Materials for RT-PCR

Dormant cuttings of 138 grapevine selections were collected from USA, Canada, Italy, and Portugal over three years. Samples included Vitis vinifera cultivars, hybrids, V. riparia, and rootstocks. 117 grapevine selections were indexed on St. George for RSP and other RW diseases. Pinot noir (1186-9A2) from Agriculture Canada, Center for Plant Health (Sidney, Canada) and Thompson seedless (RSP105) from University of California (Davis) were included as positive controls. Sauvignon blanc, generated from shoot tip tissue culture and tested free of viruses and viroids was provided by Dr. J. Semancik (University of California at Riverside) and used as a healthy control. In addition, six seedlings of five Vitis species were also included as negative controls.

Example 3 dsRNA Isolation and Analysis

Methods for isolating dsRNA were described by Hu et al., “Characterization of Closterovirus-like Particles Associated with Grapevine Leafroll Disease,” J. Phytopathology, 128:1-14 (1990), which is hereby incorporated by reference, except that 1×STE with 15% ethanol (instead of 16.5%) was used to wash CF-11 cellulose columns prior to elution of dsRNAs. The dsRNAs were isolated from leaves, petioles, and the phloem tissue of dormant canes, electrophoresed on 1% agarose or low melting temperature agarose gels, and analyzed by staining with ethidium bromide (EtBr). Hind EII digested lambda DNA was used as markers to estimate the sizes of the dsRNA molecules.

Example 4 cDNA Synthesis and Cloning

The extremely low yield of dsRNA and the limited quantity of RSP-infected grape materials precluded the use of a single RSP-infected grapevine accession as the source of dsRNA for cloning purpose. Therefore, dsRNA preparations from Colobel 257, Ravat 34, Couderc 28-112, and Seyval were pooled and used as templates for cDNA synthesis. In order to get pure templates for cloning, dsRNA bands were excised from low melting temperature agarose gels after electrophoresis and recovered by extraction with phenol and chloroform (Sambrook et al., Molecular Cloning: A LaboratorEy Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), which is hereby incorporated by reference). The same recovery procedure was repeated once more. The purified dsRNA was denatured with 20 mM methyl mercuric hydroxide and cDNAs were synthesized using slightly modified methods of Jelkmann et al., “Cloning of Four Viruses from Small Quantities of Double-Stranded RNA,” Phytopathology, 79:1250-53 (1989), which is incorporated herein be reference. The cDNA fragments were first blunt-ended with T4 DNA polymerase at 12° C. T4 DNA ligase was used to add EcoR I adapters to both ends of the cDNAs. Subsequently, the cDNA molecules with cohesive ends were ligated to EcoR I-prepared arms of lambda ZAP II. Finally, the resulting recombinant phages were packed into Gigapack II packaging extract following manufacturer's instructions (Stratagene, La Jolla, Calif.).

Example 5 Identification of cDNA Clones Specific to the dsRNA

Plaque hybridization was used to screen cDNA clones by transferring recombinant cDNA plaques to nylon membranes and hybridizing to ³²P-labeled first-strand cDNA probes generated from the dsRNA according to manufacturer's recommendations (Du Pont, 1987). Clones with strong hybridization signals were converted into pBluescript SK through in vivo excision (Stratagene, 1991). After digestion of the resulting plasmids with EcoR I, 20 clones were selected and further analyzed in Southern hybridization with radio labeled first strand cDNA probes synthesized from the dsRNA. The specificity of two selected clones to the dsRNA was confirmed by Northern analysis using ³²P labeled inserts of the two clones.

Example 6 Bridging Gaps Between Clones

To bridge the gap between clones RSP3 and RSP94, a pair of specific primers were used in RT-PCR to generate cDNA fragments from the dsRNA. RSP3-RSP94 primer 1 (sense, nt 3629-3648) has a nucleotide sequence corresponding to SEQ. ID. No. 41 as follows: GCTTCAGCAC TTGGAAGGCG 20

RSP3-RSP94 primer 2 (antisense, nt 4350-4366) has a nucleotide sequence corresponding to SEQ. ID. No. 42 as follows: CACACAGTGG CCAGCCT 17 After gel electrophoresis, PCR amplified cDNA bands were excised from gels and recovered with the phenol/chloroform method (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), which is hereby incorporated by reference).

The same strategy was employed to bridge the gap between clones RSP94 and RSP95. RSP94—RSP95 primer 1 (sense, nt 5272-5291) has a nucleotide sequence corresponding to SEQ. ID. No. 43 as follows: GGAGGTGCGT TGTGGTTATG 20

RSP94-RSP95 primer 2 (antisense, nt 6791-6808) has a nucleotide sequence corresponding to SEQ. ID. No. 44 as follows: CCGTGGCACT GCACACCC 17

Example 7 Obtaining Nucleotide Sequences on Both Termini of RSPaV-1 Genome

To obtain the terminal 3′ end sequences, a primer (sense, nt 8193-8210) having a nucleotide sequence corresponding to SEQ. ID. No. 45 as follows: GGAGGTGACC ACATTACG 18

and a (dT) 18 primer were used in RT-PCR to amplify cDNA from the dsRNA. Resulting PCR products were cloned into TA vector pCRII (Invitrogen) and sequenced. This approach was based on the assumption that the RSP associated dsRNA contained a poly (A) tail. For the terminal 5′ end, the dsRNA was first tagged with poly (A) using yeast Poly (A) polymerase (USB) (Pappu et al., “Nucleotide Sequence and Organization of Eight 3′ Open Reading Frames of the Citrus tristeza Closterovirus Genome,” Virology 199:35-46 (1994), which is hereby incorporated by reference) and then used as templates to generate cDNA fragments by RT-PCR using (dT) 18 primer and primer (antisense, nt 429-449) having a nucleotide sequence corresponding to SEQ. ID. NO. 46 as follows: CATCACGACT TGTCACAAAC C 21

Example 8 Nucleotide Sequencing

CsCl or alkaline/PEG (polyethylene glycol) purified plasmids (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), which is hereby incorporated by reference; Applied Biosystems, Inc.) and RT-PCR amplified cDNA fragments were sequenced for completion on both strands. Nucleotide sequencing was done manually with Sequenase version 2.0 kit (USB) or automatically on ABI 373 automated sequencer with Taq DyeDeoxy™ terminator cycle sequencing kit (Applied Biosystems, Inc.). Vector primers (T3, T7, M13 Forward, and M13 Reverse) were used in initial sequencing and sequences were completed by primer walking strategy.

Example 9 Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Two pairs of primers were designed for RT-PCR: (1) RSP95F1 and RSP95R1; and (2) RSP149F1 and RSP149R1. Primer RSP95F1, an antisense strand primer, has a nucleotide sequence corresponding to SEQ. ID. NO. 47 as follows: TGGGCCTCCA CTTCTTC 17

Primer RSP95R1, a sense strand primer, has a nucleotide sequence corresponding to SEQ. ID. No. 48 as follows: GGGGTTGCCT GAAGAT 16

Primer RSP149F1, an antisense strand primer, has a nucleotide sequence corresponding to SEQ. ID. No. 49 as follows: ACACCTGCTG TGAAAGC 17

Primer RSP149R1, a sense strand primer, has a nucleotide sequence corresponding to SEQ. ID. No. 50 as follows: GGCCAAGGTT CAGTTTG 17 RSP95F1/R1 were used in RT-PCR to test samples collected in 1994. RSP149R1/F1, alone or together with RSP95F1I/R1, were used to test samples collected in 1995 and 1996. To avoid bias in the judgment of RT-PCR results, blind tests were conducted for samples from Canada in 1995 and 1996. The indexing results of these samples were kept untold until the RT-PCR tests were complete.

dsRNAs were denatured with methylmercuric hydroxide (CH4HgOH) and reverse transcribed into cDNAs with Moloney murine leukemia virus (MMLV) or Avian Myeloblastosis Virus (AMV) reverse transcriptases (Promega) at 42° C. for 1 to 3 h. Five of 20 μl of the RT reactions were added to PCR mix and amplified in thermal cycler (HYBAID OmniGene, National Labnet Company) with Taq DNA polymerase (buffer B, Promega) using the following parameters: initial denaturation at 94° C. for 5 min, 40 cycles of amplification at 94° C. for 45 s, 52° C. for 1 min, and 72° C. for 1 min, and a final extension at 72° C. for 10 min. PCR products were analyzed by electrophoresis on 1% agarose gels containing ethidium bromide. Hae III digested Phix 174 fragments were used as molecular weight markers.

Example 10 Southern Blot

DNA fragments amplified by PCR from cDNA clone RSP149 with primers RSP149F1/R1 were labeled with ³²P by random priming and used as probes. Products of RT-PCR of randomly selected grapevines including 26 positives and 6 negatives by RT-PCR were electrophoresed on an 0.8% agarose gel, transferred to nylon membranes, and hybridized to the probes following manufacturer's instructions (Du Pont).

Example 11 Computer Assisted Analysis of Sequences and Genome Structure of RSPaV-1

Sequences were assembled with SeqMan program and potential open reading frames were generated with MapDraw program (DNASTAR, Madison, Wis.). BLAST program of the NCBI (the National Center for Biotechnology Information) was used to search for homologies in DNA and protein databases. Clustal analysis (with identity weight table) of MegAlign (DNASTAR) was employed to reveal sequence similarities between the putative proteins of RSPaV-1 and the analogous proteins of ASPV (Jelkmann, “Nucleotide Sequences of Apple Stem Pitting Virus and of the Coat Protein of a Similar Virus from Pear Associated with Vein Yellows Disease and Their Relationship with Potex- and Carlaviruses,” J. General Virology, 75:1535-42 (1994), which is hereby incorporated by reference) and PVM (Zavriev et al., “Complete Nucleotide Sequence of Genomic RNA of the Potato M-Virus,” Molecular Biology (Mosk.) 25:761-69 (1991), which is hereby incorporated by reference). In addition, nucleotide sequences of the untranslated regions (UTR) of these three viruses were also compared using MagAlign, as shown in FIGS. 6A and 6B.

Example 12 Consistent Association of a High Molecular Weight dsRNA with RSP

The 15 grapevine accessions used in this study were previously indexed on St. George where 12 accessions induced typical RSP symptoms (i.e., a narrow strip of small pits below the inoculum bud). FIG. 1A illustrates these typical RSP symptoms. A good correlation was found between the presence of the specific dsRNA and the indexing results on St. George. As shown in FIG. 2A and recorded in Table 1 below, twelve grapevine accessions with typical RSP symptoms revealed a dsRNA of ca. 8.7 kb with gel electrophoresis. In addition, a smaller dsRNA of about 6.6 kb was observed in Colobel 257 and Seyval. In contrast, although Aminia and Canandaigua elicited deep pits and grooves around the woody cylinder of St. George, they did not reveal visible dsRNA of expected size in repeated experiments. Freedom, which indexed negative for RSP on St.

George, did not reveal visible dsRNA. Although two dsRNA bands were observed in Verduzzo 233A (which was indexed free of RSP on St. George), they were not specific to RSP based on the fact that they were larger or smaller than the 8.7 kb dsRNA associated with RSP (FIG. 2A) and that they did not hybridize to the RSP-specific probe in Northern analysis (FIG. 2B). In addition, the two dsRNA species isolated from Verduzzo 233A were not observed in other healthy grapevines such as Cabernet Franc and LN 33. TABLE 1 St. George Accessions and Parentage Indicator dsRNA Northern Aminia (Carter X Black Hamburg) + − − Bertille Seyve 3408 (BS 872 X + + + Seibel 5410) Bertille Seyve 5563 (Seibel 6905 + + + X BS 3445) Canandaigua (V. labrusca X + − − V. vinifera) Colobel 257 (Seibel 6150 X + + + Seibel 5455) Couderc 28-112 (Emily X V. rupestris) + + + Freedom (Couderc 1613 X Dog Ridge) − − − Grande Glabre (V. riparia) + + + Ill 344-1 (BS 2667 X Seibel 6905) + +^(†) −^(†) Joffre (V. vinifera X V. riparia + + + X V. rupestris) Ravat 34 (Berlandieri X Chardonnay) + + + Seyval (Seibel 4995 X Seibel 4986) + + + Seyve Villard 14-287 (V. labrusca + + + X V. rupestris X V. aestiv X V. cinerea X V. vinifera) Seyve Villard 3160 (Seibel 5163 + + + X Seibel 2049) Verdelet (Seibel 5455 X Seibel 4938) + + + Controls Pinot Noir (V. vinifera) + + + Thompson seedless (V. vinifera) + NT + Verduzzo 233A − −^(‡) − Symbols: *Probe used was insert from cDNA clone RSP149. ^(†)A faint dsRNA band could be observed on the gel after electrophoresis but no hybridization signal could be seen in Northern analysis. ^(‡)Although two dsRNA bands were observed in Verduzzo 233A, they were not specific to RSP, because they were either larger or smaller than the RSP-associated 8.7 kbp dsRNA and they did not hybridize to the probe in Northern analysis.

The yield of dsRNA was low and varied significantly among different accessions. When a comparable amount of phloem tissue (14 g for Bertille Seyve 5563 and Couderc 28-112; 18.5 g for the others) was used to isolate dsRNA, Colobel 257, Seyval, Ravat 34, Grande Glabre, and Seyve Villard 14-287 displayed strong dsRNA bands, while Bertille Seyve 5563, Couderc 28-112, Joffre, and Verdelet showed weak bands after staining with EtBr, as shown in FIG. 2A. Bertille Seyve 3408 and Seyve Villard 3160 were analyzed in separate experiments and dsRNA bands of the same size were observed.

Example 13 Selection and Specificity of cDNA Clones

A total of 182 clones were selected after plaque hybridization. Eighty clones with strong hybridization signals were subcloned into pBluescript SK through in vivo excision. Resulting plasmids were shown to have inserts ranging from 0.3 to 3.0 kb. A total of 20 clones with inserts of ca. 0.8 kb or larger were selected. Southern analysis of these 20 clones to radio labeled first strand cDNA probes derived from the dsRNA resulted in 15 clones with strong hybridization signals. Several of these clones were used to determine the genome sequence of the dsRNA: RSP3, RSP28, RSP94, RSP140, RSP95, and TA5. Another clone (RSP149), which was 97% similar in nucleotide sequence to RSP95, was used as one of the two probes in Northern hybridization.

Northern hybridization was employed to confirm the specific relationship of clones RSP95 and RSP149 to the isolated dsRNA. These two clones gave the strongest reaction in Southern analysis described above. Initial experiments showed that RSP95 insert hybridized with the dsRNA isolated from three accessions (Colobel 257, Seyval, and Ravat 34), from which the template dsRNAs used in cDNA synthesis were isolated. As shown in FIG. 2B and indicated in Table 1, use of RSP 149 insert as the probe showed that this clone hybridized with the dsRNA of ca. 8.7 kb isolated from RSP infected grapevines. Furthermore, the intensity of hybridization signals corresponded to that of the dsRNA bands observed on agarose gels stained with EtBr. Colobel 257, Seyval, Ravat 34, Grande Glabre, and Serve Villard 14-287 reacted strongly; Bertille Seyve 5563, Couderc 28-112, Joffre, and Verdelet had weak hybridization signals. The result for Ill 344-1 was not conclusive. Aminia and Canandaigua did not show visible dsRNAs or hybridization in Northern analysis. Bertille Seyve 3408, which was tested in a separate experiment, did show a ca. 8.7 kb dsRNA which hybridized to the probe from RSP149. Freedom and Verduzzo 233A, which had indexed negative for RSP on St. George, were also negative in Northern blot.

Example 14 Nucleotide Sequence and Genome Structure of RSPaV-1

Six cDNA clones and three RT-PCR amplified cDNA fragments (identified as RSPA, RSPB, and RSPC) were sequenced on both strands and used to obtain the complete nucleotide sequence of a viral agent, which is shown in FIG. 3A. The genome of RSPaV-1 consisted of 8726 nts excluding a poly (A) tail on the 3′ end. The sequence of RSPA indicated that the 5′ first base of the RSPaV-1 genome appeared to be a cytosine (C). Clone TA5, which represented the 3′ end of the RSPaV-1 genome, contained a stretch of adenines (A) preceded by a cytosine.

MapDraw analysis, shown at FIG. 3B, indicated that the genome of RSPaV-1 had five potential ORFs on its positive strand, while no ORFs were observed on the negative strand (data not shown). ORF1 (nt 62 to 6547 of SEQ. ID. No. 1) has a nucleotide sequence corresponding to SEQ. ID. NO. 2. ORF1 believed to encode a protein or polypeptide having a molecular weight of about 244 kDa and an amino acid sequence corresponding to SEQ. ID. No. 3. According to Lutcke et al., “Selection of AUG Initiation Codons Differs in Plants and Animals,” Eur. Mol. Biol. J., 6:43-48 (1987), which is hereby incorporated by reference, the start codon of ORF1 was in a favorable context: GCAAUGGC, where the “GC” after the start codon is important for initiating translation in a plant system. ORF2 (nt 6578 to 7243 of SEQ. ID. No. 1) has a nucleotide sequence corresponding to SEQ. ID. No. 4. ORF2 is believed to encode a protein or polypeptide having a molecular weight of about 24.4 kDa and an amino acid sequence corresponding to SEQ. ID. NO. 5. The first two ORFs were separated by an intergenic region of 30 nts. ORF3 (nt 7245 to 7598 of SEQ. ID. NO. 1) has a nucleotide sequence corresponding to SEQ. ID. No. 6. ORF3 is believed to encode a protein or polypeptide having a molecular weight of about 12.8 kDa and an amino acid sequence corresponding to SEQ. ID. NO. 7. ORF4 (nt 7519 to 7761 of SEQ. ID. NO. 1), which overlapped with ORF3 by 80 nts, has a nucleotide sequence corresponding to SEQ. ID. No. 8. ORF3 is believed to encode a protein or polypeptide having a molecular weight of about 8.4 kDa and an amino acid sequence corresponding to SEQ. ID. No. 9. Nine nucleotides downstream of ORF4 was the start of ORF5 (nt 7771 to 8550 of SEQ. ID. No. 1), which has a nucleotide sequence corresponding to SEQ. ID. No. 10. ORF5 is believed to encode a protein or polypeptide having a molecular weight of about 28 kDa and an amino acid sequence corresponding to SEQ. ID. No. 11. Downstream of ORF5 was the 3′ end LJTR of 176 nts. Although computer assisted analysis indicated that two shorter ORFs may exist as alternatives to ORF1 and ORF5, neither of them were in good contexts for translation initiation.

Example 15 Comparison of the RSPaV-1 Genome with ASPV and PVM Carlavirus Genomes

The arrangement of the ORFs and the amino acid sequences of RSPaV-1 showed similarities to those of PVX (Skryabin et al., “The Nucleotide Sequence of Potato Virus X RNA,” Nucleic Acids Res. 16: 10929-30 (1988), which is hereby incorporated by reference), PVM (Zavriev et al., “Complete Nucleotide Sequence of Genomic RNA of the Potato M-Virus,” Molecular Biology (Mosk.) 25:761-69 (1991), which is hereby incorporated by reference), and ASPV (Jelkmann, “Nucleotide Sequences of Apple Stem Pitting Virus and of the Coat Protein of a Similar Virus from Pear Associated with Vein Yellows Disease and Their Relationship with Potex- and Carlaviruses,” J. General Virology 75:1535-42 (1994), which is hereby incorporated by reference), with the latter two being the most similar to RSPaV-1. A representation of the sequence comparison is shown in FIG. 3B and the percent identities in amino acid sequences of the ORF of RSPaV-1 and the corresponding ORF of ASPV, PVM, and PVX are shown in Table 2 below. These analyses suggest that the ORFs of RSPaV-1 are compared with those of PVM and ASPV. TABLE 2 Replicase Coat ORF1 Protein Region I Region II Triple Gene Block ORF5 aa 1-372 aa 1354-2161 Total ORF2 ORF3 ORF4 Total aa 142-245 ASPV 49.2 57.5 39.6 38.0 39.3 27.1 31.3 49.5 PVM 47.2 53.2 37.6 34.8 31.2 19.0 21.2 33.3 PVX 18.9 20.4 15.7 23.5 31.3 22.9 27.4 42.9

When the total amino acid sequence of RSPaV-1 ORF 1 was used for comparison, it showed 39.6% and 37.6% identities with the replicases of ASPV and PVM respectively (Table 2). These homologies were mainly found in regions I (aa 1 to 372) and II (aa 1354-2161), which are at the N and C terminal portions of the putative replicase, respectively, shown at FIGS. 4A and 4B. Within region I, the identities of RSPaV-1 with ASPV and PVM were 49.2% and 47.2%, respectively (Table 2). The methyltransferase domain, which is conserved in Sindbis-like superfamily of plant viruses (Rozanov et al., “Conservation of the Putative Methyltransferase Domain: A Hallmark of the “Sindbis-like” Supergroup of Positive-Strand RNA Viruses,” J. General Virology 73:2129-34 (1992), which is hereby incorporated by reference), was found in this region (FIG. 4A). Region II, on the other hand, showed even higher identities: 57.5% with ASPV and 53.2% with PVM (Table 2). A NTP binding motif “GXXXXGKS/T” (aa 1356 to 1363) (“X” stands for any amino acid residue), which is conserved in helicase proteins and helicase domains of eukaryotic positive strand RNA viruses (Gorbalenya et al., “A Novel Superfamily of Nucleotide Triphosphate-Binding Motif Containing Proteins which are Probably Involved in Duplex Unwinding in DNA and RNA Replication and Recombination,” FEBS Letters, 235:16-24 (1988), which is hereby incorporated by reference), was found in the beginning of region II (FIG. 4B). The amino acid sequences of this motif in ASPV and PVM were identical to that of RSPaV-1 except for one position. Furthermore, amino acid sequence surrounding the GDD motif, which is conserved in all RNA dependent RNA polymerases of positive strand RNA viruses (Koonin, “The Phylogeny of RNA-Dependent RNA Polymerases of Positive-Strand RNA Viruses,” J. Gen. Virology 72:2197-2206 (1991), which is hereby incorporated by reference), was located near the C terminus of the RSPaV-1 replicase protein and showed high identities to those of ASPV and PVM (FIG. 4B). Other conserved residues of positive strand RNA viruses as described by Koonin, “The Phylogeny of RNA-Dependent RNA Polymerases of Positive-Strand RNA Viruses,” J. Gen. Virology 72:2197-2206 (1991), which is hereby incorporated by reference, were also found in this region. Based on these information, it was concluded that ORF1 of RSPaV-1 codes for the putative replicase protein.

The triple gene block is a common feature of several groups of plant viruses including carlaviruses, potexviruses, and ASPV. Comparison of RSPaV-1 ORF2 with those of PVM and ASPV showed evenly distributed homologies in amino acid sequence: 38.0% identity to ASPV and 34.8% to PVM (Table 2). The N terminal region of the 24.4K protein (ORF2) contained the consensus sequence “GXGKS S/T” (aa 31 to 36) (FIG. 5A), which is observed in its counterparts in carlaviruses (Zavriev et al., “Complete Nucleotide Sequence of Genomic RNA of the Potato M-Virus,” Molecular Biology (Mosk.) 25:761-69 (1991), which is hereby incorporated by reference) and a number of ATP and GTP binding proteins (Zimmem, “Evolution of RNA Viruses,” in RNA Genetics, Holland et al., eds., CRC Press, Boca Raton, Fla., USA (1987), which is hereby incorporated by reference). The 12.8K protein of RSPaV-1 encoded by ORF3 had 39.3% and 31.2% identities with its counterparts in ASPV and PVM respectively (Table 2). However, most of the matching occurred in a region from aa 29 to 62, among which 18 aa were fully conserved in all three viruses (FIG. 5B). These 12-13K proteins may function in membrane binding (Morozov et al., “Nucleotide Sequence of the Open Reading Frames Adjacent to the Coat Protein in Potato Virus X Genome,” FEBS Letters 213:438-42 (1987), which is hereby incorporated by reference). The 8.4K protein encoded by RSPaV-1 ORF4, in contrast, showed much lower identities: 27.1% with that of ASPV and 19.0% with that of PVM (Table 2). However, four residues “TGES” (aa 38 to 41) were conserved in all three viruses (FIG. 5C). In vitro studies indicated that the analogous 7K protein of PVM may bind to single or double stranded nucleic acids (Gramstat et al., “The 12 kDa Protein of Potato Virus M Displays Properties of a Nucleic Acid-Binding Regulatory Protein,” FEBS Letters, 276:34-38 (1990), which is hereby incorporated by reference) and to plasma membrane (Morozov et al., “In vitro Membrane Binding of the Translation Products of the Carlavirus 7-kDa Protein Genes,” Virology 183:782-85 (1991), which is hereby incorporated by reference).

A sequence similarity search in a DNA database revealed identities between the putative protein encoded for by RSPaV-1 ORF5 to the coat proteins (CPs) of several groups of plant viruses, indicating that RSPaV-1 ORF5 may code for the coat protein. MegAlign analysis revealed that RSPaV-1 ORF5 had 31.3% and 21.2% identities with the CPs of ASPV and PVM, respectively (Table 2). Most of the identities were found in the C terminal portion of the coat proteins (aa 142 to 245 for RSPaV-1), while the N terminal portions were quite variable in the numbers and sequences of amino acid residues. When the C terminal portion of RSPaV-1 CP was compared to the corresponding regions of ASPV and PVM, it showed 49.5% and 33.3% identities with ASPV and PVM, respectively (Table 2). In addition, the “RR/QX-XFDF” motif was found in the central region of RSPaV-1 CP (FIG. 5D). This motif is conserved in the CPs of positive strand RNA viruses with filamentous morphology and were reported to be involved in salt bridge formation (Dolja et al., “Phylogeny of Capsid Proteins of Rod-Shaped and Filamentous RNA Plant Virus: Two Families with Distinct Patterns of Sequence and Probably Structure Conservation,” Virology, 184:79-86 (1991), which is hereby incorporated by reference). Therefore, it is believed that ORF5 encodes a putative coat protein.

MegAlign analysis, shown in FIGS. 6A and 6B, revealed that the 3′ UTR of RSPaV-1 is more similar to that of PVM than to that of ASPV. For example, in a 75 nts stretch, RSPaV-1 had 68% identity with PVM. Within this region, 21 consecutive nucleotides were identical between these two viruses. The significance of this conservation in nucleotide sequence remains to be explored. In contrast, the 5′ UTR of RSPaV-1 did not reveal significant similarities with those of PVM and ASPV.

It has been have shown that an 8.7 kbp dsRNA is consistently associated with grapevines that indexed positively on St. George for RSP. Sequence analyses of the dsRNA provide evidence that a virus is involved in RSP, which has now been named RSPaV-1. The complete nucleotide sequence of RSPaV-1 was determined from overlapping cDNA clones and RT-PCR-amplified cDNA fragments generated from the dsRNA. The RSPaV-1 genome has five ORFs coding for the putative replicase (ORF1), the triple gene block (ORF24), and the CP(ORF5). The existence of these ORFs and their potential to code for structural and non-structural viral proteins were further supported by the identification of conserved motifs which are the signatures of various viral proteins.

This work confirms and extends the findings of Walter and Cameron (“Double-stranded RNA Isolated from Grapevines Affected by Rupestris Stem Pitting Disease,” Am. J. Enology and Viticulture 42:175-79 (1991), which is hereby incorporated by reference), and Azzam and Gonsalves (“Detection of dsRNA in Grapevines Showing Symptoms of Rupestris Stem Pitting Disease and the Variabilities Encountered,” Plant Disease 75:960-64 (1991), which is hereby incorporated by reference), who observed a major dsRNA species of about 8.0-8.3 kbp in RSP-infected grapevines. In addition, such work also observed a smaller dsRNA of ca. 6.6 kbp. A dsRNA of similar size was also observed here, but it was consistently detected in only Colobel 257 and Seyval. The relationship, if any, of this smaller dsRNA to RSP remains to be determined. The small dsRNA of ca. 0.359 kbp, which Monette et al. (“Double-stranded RNA from Rupestris Stem Pitting-Affected Grapevines,” Vitis 28:13744 (1989), which is hereby incorporated by reference) isolated from RSP-infected grapevines growing in tissue culture, was not observed.

Electron microscopy evidence also suggests that RSP is caused by filamentous virus(es). Tzeng et al. (“Anatomical and Tissue Culture Studies of Rupestris Stem Pitting-Affected Grapevines,” Botan. Bulletin of Acad. Sinica (Taipei) 34:73-82 (1993), which is hereby incorporated by reference) observed flexuous filamentous virus aggregates in the phloem parenchyma cells of young shoots of Sylvner grapevines that had indexed positively for RSP. Monette and Godkin (“Detection of Capillovirus-like Particles in a Grapevine Affected with Rugose Wood,” Vitis 34:24142 (1995), which is hereby incorporated by reference) observed a filamentous virus in Sauvignon blanc infected by RSP and LNSG. The relationship of these virus particles to RSP disease remains to be studied.

Evidence suggests that the cDNA library generated from the isolated dsRNA templates is not homogeneous for only RSPaV-1. During the process of sequencing cDNA clones, several clones (e.g., RSP474 and RSP158) were identified with high, but not identical, sequence similarities to RSPaV-1.

RSPaV-1 has the most similarities to ASPV, which has not yet been grouped into a virus genus. Both viruses have the same genome organization and their ORFs code for putative proteins of similar sizes, except that the coat protein of ASPV is significantly larger (44 kDa) than that of RSPaV-1 (28 kDa). Comparisons of RSPaV-1 with PVM carlavirus show some similarities in genome organization except that RSPaV-1 lacks ORF6 which is located at the 3′ end of PVM genome. Although the genome organization of RSPaV-1 is similar to PVX potexvirus, the latter has a much smaller putative replicase. RSPaV-1 has no relation to grape viruses whose genomes have been sequenced so far. The closest possibilities, GVA (Minafra et al., “Grapevine virus A: Nucleotide Sequence, Genome Organization, and Relationship in the Trichovirus Genus,” Arch. Virology 142:417-23 (1997), which is hereby incorporated by reference) and GVB (Saldarelli et al., “The Nucleotide Sequence and Genomic Organization of Grapevine Virus B,” J. General Virology 77:2645-52 (1996), which is hereby incorporated by reference), have different genome structures than RSPaV-1.

Example 16 Specific and Universal Primers and the Detection of Different Strains of RSPaV by Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Among the 138 grapevine entries collected, 25 indexed negatively and 93 indexed positively for RSP on St. George, while the others were not indexed (see Tables 3-7 below). Symptoms induced by RSP on the woody cylinder of St. George after graft inoculation with chip-buds can be divided into two types. The first type is called “specific”, that is, pits and/or grooves being restricted to the area on the woody cylinder below the inoculation sites. The other is called “nonspecific”, that is, pits and/or grooves being present above, around, and below the inoculation sites. TABLE 3 Cultivar/Accession ID Index St.G RT-PCR Source Almeria K3 P 661 1483-13D1 − − C Auxerrois CL 56 658-1A2 − −a C Auxerrois CL 56 658-1A1-1A2 − − C GM 32458 604-8A2-2A2 − − C GM 7117-10 1347-16A1 − −a C Italia 1186-5B1 − − C Pslanka (H) 23-10A2-2A2 − − C Ventura (V. 51061) 1166-2A1 − − C (H) Verdelet (H) 1170-3C2-2S1 − − C Verduzzo (V) 233A − − I Vivant (V. 63331) (H) 1166-3A1 − − C Control Sauvignon Blanc (V) AV-4 #2 − −a U Symbols: V., Vitis vinefera; R., Vitis riparia; H., hybrid; C., Canada; I., Italy; U., USA; P., Portugal; a, tested by RSP149F1/R1 and 95F1/R1 and results agree to each other; b, tested by 95F1/R1 only

TABLE 4 Index Cultivar/Accession ID St.G RT-PCR Source Aragonez (Temperanillo) 238 − + P Albalonga 1058-4A2-2A1 − + C Cabernet Franc (V) 147A − + I Chardonnay (V) 80A − + I Ehrenfelser PM 1 (V) 1169-1A1 − + C Freedom (H) PI 588370 − +a U Harslevellu P 679 1483-2B1 − + C Heroldrebe 1318-2A1 − + C Malvasia Fina 340 − + P Perle of Zala 1407-5A1 − + C Refosco (V) 181A − + I San Giovese 1497-2A1 − + C Brunello CL BBS 11 Touriga Francesa 313 − + P Symbols: V., Vitis vinefera; R., Vitis riparia; H., hybrid; C., Canada; I., Italy; U., USA; P., Portugal; a, tested by RSP149F1/R1 and 95F1/R1 and results agree to each other; b, tested by 95F1/R1 only

TABLE 5 Index RT- Cultivar/Accession ID St.G PCR Source Albalonga 1058-4A2-1A2 + + C Aminia (H) PI 588306 + + U Antao Vaz CL 245 + + P Aragonez (Temperanillo) 350 + + P Auxerrois CL 56 658-1A1 + + C Badacsony-10 1407-1A1 + + C Bertille Seyve 3408 (H) GVIT 348 + +b U Bertille Seyve 5563 (H) PI 181647 + +a U Blauer Spatburgunder Q1378-1 + +b C Blauer Zwiegelt/5BB 1240-1A1 + +a C Bonbino B 9 1586-17P3 + + C Brant (H) 1078-1A1 + + C Cabernet Franc (V) 151A + + I Cabernet Sauvignon (V) 124A + + I Cardinal Q390-13 + +b C Chardonnay (V) Q661-4 + +b C Chardonnay CL 116 (V) 1021-13A2 + +a C Chardonnay (V) 128B + +b I Chardonnay (V) 72A + +b I Chardonnay (V) 73A + +b I Chardonnay (V) 83A + + I Chazan CL 538 1346-6A1 + +a C Chenin Blanc CL 220 1555-6A1 + + C Colobel 257 (Seibel 8357) (H) PI 588062 + +a U Couderc 28-112 (H) PI 588248 + +a U De Chaunac S9549 (H) Q659-1 + +b C Durella 3 1586-13P1 + + C Esgana cao 276 + + P Egri Csillagok-30 1407-3A1 + + C Gamay Precoce 1500-2A1 + + C GM 31875 782-18A1 + +a C GM 32458 604-8A1 + + C GM 32458 782-21B1 + + C GM 6417-7 1347-7A1 + + C GM 6497-4 1347-14A1 + + C GM 7116-10 1362-4A1 + + C GM 7117-13 1347-17A2 + + C Grande Glabre (R) 279897 + +a U Gyongyriziling 1407-4A1 + + C ILL 344-1 (H) GVIT 658 + +a U Joffre (Kuhlmann 187-1) (H) GVIT 381 + +a U Koret (H) Q1179-7 + +b C Malvasia (V) 153A + + I Malvasia (V) 161A + + I Merlot CL 447 (V) 1236-17A1 + + C Moureto 87 + + P Moureto 96 + + P Muscat De Hambourg CL 202 1346-5A1 + + C Perle of Csaba Q806-1 + +b C Pinot Chardonnay CL 76 (V) 949-3A2 + +a C Pinot Chardonny CL 277 (V) 949-8B1 + + C Pinot Grigio (V) 104A + +b I Pinot Grigio (V) 108A + +b I Pinot Grigio (V) 114A + + I Pollux B6-18 1357-4A1 + + C Pslanka (H) 23-10A2 + + C Ravat 34 PI 588247 + +a U Refosco (V) 190A + +? I Refosco (V) 195A + + I Riesling CL 49 (V) 1555-2A1 + +a C San Giovese Brunello 1497-3B1 + + C CL E BS 4 Schew-Rebe 778-6A1 + +a C Semillon CL 299 (V) 1555-7A1 + +a C Seyval Blanc PI 588309 + +a U (Seyve Villard 5-276) (H) Seyve Villard 14-287 (H) PI 588246 + +a U Seyve Villard 3160 (H) PI 181630 + +a U Titan Q1235-1 + +b C Verdelet (H) PI 186260 + +a U Verdelho 274 + + P Verduzzo (V) 222A + +b I Verduzzo (V) 226A + +b I Verduzzo (V) 239A + + I Vidal Blanc 1200-5A1 + +a C Weiser Burgunder Q782-40 + +b C 3309 C 330-4A1 + + C 420 A 1483-4A1 + + C 7542 Q1386-1 + +b C Pinot Noir (V) 1186-9A2 + +a C Thompson Seedless (V) RSP105 + +a U Symbols: V., Vitis vinefera; R., Vitis riparia; H., hybrid; C., Canada; I., Italy; U., USA; P., Portugal; a, tested by RSP149F1/R1 and 95F1/R1 and results agree to each other; b, tested by 95F1/R1 only.

TABLE 6 Index Cultivar/Accession ID St.G RT-PCR Source Aligote Q637-2B2 + −b C Aragonez (Temperanillo) 232 + − P Canandaigua (H) GVIT 566 + −a U Challenger (H) Q1338-1 + −b C Fercal CL 242 1551-4A1 + −a C GM 7746-6 1362-6A1 + − C Gravesac CL 264 1551-3A1 + −a C Honey Red 1339-6A1 + − C Kee-Wah-Din (H) 1278-1A1 + − C Periquita 72 + − P Tajoznyt Izumrud (H) Q2-2 + −b C Thurling 1047-4A2-1A2 + − C Verdelet 1170-3D2-2A1 + − C 5BB CL 114 1236-2A1 + − C Alphonse Lavalle NI + I Ancellotta NI + I Chardonnay (V) 127 NI + I Kober 5BB? 100 NI + I Moscato d'Adda 7 NI + I Periquita 624 NI + P Periquita 633 NI + P Riesling (V) 3 NI + I Seyval (H) Peterson NI + U Terrano 1/1/3/K NI + I Thurling 1047-4A2-2A1 NI − C Tocai Rosso 19 1586-21P4 NI + C Trebbiano Toscano 67 NI − I Vidal Peterson NI + U Symbols: V., Vitis vinefera; R., Vitis riparia; H., hybrid; NI, not indexed; C., Canada; I., Italy; U., USA; P., Portugal; a, tested by RSP149F1/R1 and 95F1/R1 and results agree to each other; b, tested by 95F1/R1 only

TABLE 7 Cultivar/Accession ID Index St.G RT-PCR Source V. acerifolia PI 588448 NI − U V. acerifolia PI 588449 NI − U V. cinerea PI 588446 NI − U V. monticola PI 588454 NI − U V. riparia PI 495622 NI − U V. sp. yenshanesis PI 588421 NI − U Symbols: V., Vitis vinefera; R., Vitis riparia; H., hybrid; NI, not indexed; C., Canada; I., Italy; U., USA; P., Portugal; a, tested by RSP149F1/R1 and 95F1/R1 and results agree to each other; b, tested by 95F1/R1 only

Among the 93 RSP-infected grapevines, 79 (85%) produced cDNA fragments of expected sizes in repeated RT-PCR using RSP149F1/R1 primers (SEQ. ID. Nos. 49 and 50) and/or RSP95F1/R1 primers (SEQ. ID. Nos. 47 and 48), while the other 14 were negative (see Tables 5 and 6). Interestingly, 12 of 14 (85.7%) grapevine accessions which were not indexed for RSP also produced cDNA fragments of expected size in RT-PCR (see Table 6). Sauvignon blanc healthy control) was negative in repeated RT-PCR (see Table 3).

Results of RT-PCR for grapevines indexed negatively for RSP were surprising (see Tables 3 and 4). While 11 were negative in RT-PCR tests (excluding Sauvignon blanc healthy control), the other 13 produced cDNA fragments of expected sizes.

Since RSPaV-1 was detected not only from grapevines which indexed positively for RSP but also from some of the grapevines indexed negatively for RSP, a search for more healthy materials for RT-PCR tests became necessary. As the majority of plant viruses do not pass on through seeds, grapevine seedlings are probably free of RSPaV-1. Based on this assumption, six seedlings from five Vitis species were included in RT-PCR (see Table 7). None of them produce cDNA of expected size in RT-PCR using RSP149R1/F1 primers (SEQ. ID. Nos. 49 and 50).

The data described above (and shown in Tables 3-7) indicate that RSPaV-1 is closely associated with RSP and that it is likely the causal agent of RSP. RT-PCR detected RSPaV-1 specific sequences from most of the RSP-infected grapevines collected from a wide range of viticultural regions of the world. Among the 93 grapevine accessions indexed positively for RSP on St. George, 85% were positive in RT-PCR (see Table 5). The data also suggests that RT-PCR has the potential to be used as a standard method for diagnosing RSP. This method is advantageous over the biological indexing on indicator St. George, because it is simpler, quicker, and more sensitive.

RT-PCR did not detect RSPaV-1 sequences from 14 of the grapevine accessions indexed positively for RSP (see Table 6). The discrepancy between RT-PCR and indicator indexing can be attributed to the existence in grapevines of different viruses or strains of the same virus which may all induce similar pitting and/or grooving symptoms on St. George upon graft-inoculation. It is believed these agents are only slightly different from RSPaV-1 at the level of their nucleotide sequences, but significant enough to hinder them from being detected by RT-PCR using RSPaV-1 specific primers.

It is likely that many RSPaV strains have genomes with nucleotide sequences that are highly similar to the nucleotide sequence of the RSPaV-1 genome. Evidence that supports this hypothesis includes the finding of a highly conserved region of ca. 600 bps among the nucleotide sequences of RSPaV-1 (type strain) and seven other cDNA clones, as shown in FIG. 9. The nucleotide sequence identities of these strains to RSPaV-1 (type strain) range from 83.6% to 98.4%. If oligonucleotides are chosen which are conserved among all these strains (i.e., with one or only a few mismatches), then the oligonucleotides should function as universal primers, allowing all of the strains to be detected by RT-PCR. Based on this theory, a primer pair (BM98-3F/BM98-3R) can be designed to amplify a DNA fragment of 320 bps from all these clones. BM98-3F has a nucleotide sequence corresponding to SEQ. ID. No. 51 as follows: GATGAGGTCCAGTTGTTTCC 20

BM98-3R has a nucleotide sequence corresponding to SEQ. ID. No. 52 as follows: ATCCAAAGGACCTTTTGACC 20 Primers BM98-3F/BM98-3R can be used in RT-PCR to test further some of the grapevine samples which were negative for RSPaV in RT-PCR using RSP95F1/RSP95R1 primers (SEQ. ID. Nos. 47 and 48, respectively) or RSP149F1/RSP149R1 primers (SEQ. ID. Nos. 49 and 50, respectively). Results show that 6 of the 9 samples included were positive for RSPaV in RT-PCR using BM98-3F/BM98-3R primers. This indicates that these universal primers can be used to achieve even higher detection rates.

Another pair of primers (BM98-1F/BM98-1R) can be designed in a way that they can amplify DNA of 760 bps from RSPaV-1, RSP47-4, and RSP158. BM98-1F has a nucleotide sequence corresponding to SEQ. ID. No. 53 as follows: CTTGATGAGTACTTGTC 17

BM98-1R has a nucleotide sequence corresponding to SEQ. ID. No. 54 as follows: GCAAGGATTTGGATGGC 17 Other “universal primers” can be designed manually or with computer programs (such as PrimerSelect) in the same way so that they contain conserved regions of nucleotide sequences for different strains of RSPaV-1.

RT-PCR detected RSPaV-1 sequences from 54% of grapevines negative for RSP as judged by indexing on St. George (see Tables 3 and 4). Several possibilities may account for this discrepancy. First, RT-PCR is much more sensitive than indicator indexing. Virus(es) of extremely low concentration may not induce visible symptoms on St. George within the standard indexing period, while they can be detected by RT-PCR. Second, judging indexing results can, in some cases, be very subjective. For example, it is very difficult to reach a conclusion on whether a grapevine is infected with RSP when only one or a few small pits are present on the woody cylinder of St. George. Third, uneven distribution of virus(es) within grapevines and the relatively limited number of replicates of St. George indicators may result in the failure to detect RSP-infection.

RSP seems to be widespread in different types of grapevines including V. vinifera, hybrids, V. riparia, and rootstocks. It occurs in a wide range of geographic regions including North America, Europe, Australia, and possibly many other countries as well. Testing grapevines from other areas of the world using RSPaV-1 specific primers will provide definitive information on the exact distribution of RSP throughout the world. It is also interesting to investigate whether RSP is transmitted by any vectors in nature.

RSP is a disease under quarantine in Washington and New York of the USA. Since this work and the work of others (Golino and Butler, “A Preliminary Analysis of Grapevine Indexing Records at Davis, California,” in Proceedings of the 10th Meeting of the ICVG, pp. 369-72, Rumbos et al., eds., Volos, Greece (1990); Azzam and Gonsalves, “Detection of dsRNA in Grapevines Showing Symptoms of Rupestris Stem Pitting Disease and the Variabilities Encountered,” Plant Disease, 75:96-964 (1991); Garau, “Kober Stem Grooving and Grapevine Virus A: A Possible Relationship,” in Extended Abstracts of the 11th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine, p. 54, Montreux, Switzerland (1993); Credi, “Characterization of Grapevine Rugose Wood Sources from Italy,” Plant Disease, 82:1288-92 (1997), all of which are hereby incorporated by reference) showed that RSP is so wide-spread, it is questionable whether or not RSP should be kept under plant quarantine any longer. The devlopment and advance of rapid diagnostic methods will also allow us to investigate on the economic damage caused by RSP.

According to Goheen (“Rupestris Stem Pitting,” in Compendium of Grape Diseases, p. 53, Pearson and Goheen, eds., American Phytopathological Society Press, St. Paul, Minn., USA (1988), which is hereby incorporated by reference), RSP is a disease which induces, after graft-inoculation with a chip bud from an infected grapevine, a row of small pits on the woody cylinder of St. George below the point of inoculation. This definition may not be comprehensive. Indexing record indicated that two types of stem pitting (specific vs. nonspecific) were often observed on the woody cylinder of St. George upon graft inoculation with chip buds. For example, among 16 RSP-positive grapevines collected from Canada in 1995, eight developed specific type symptoms, while the others produced nonspecific symptoms. Credi (“Characterization of Grapevine Rugose Wood Sources from Italy,” Plant Disease, 82:1288-92 (1997), which is hereby incorporated by reference) also observed these two types of stem pitting in his indexing work. However, from the primers used in RT-PCR, as described above, RSPaV-1 was detected in grapevines showing both types of symptoms on St. George.

Thus, RT-PCR detected RSPaV-1 sequences from a wide range of grapevines collected from a number of major grapevine growing countries. The data clearly suggest that RSPaV-1 is closely associated with Rupestris stem pitting of grapevines and that it is likely the causal virus of RSP. Use of “universal” primers which can detect multiple agents which are highly similar to RSPaV-1 in nucleotide sequences would improve the detection rate by RT-PCR In addition, antibodies produced against bacteria-expressed coat proteins of RSPaV-1 will help in finding the viral particles from RSP infected grapevines and in rapid detection of RSP.

Example 17 Southern Hybridization

To confirm the specificity of the RT-PCR products to RSPaV-1, Southern blot hybridization was conducted using ³²P labeled probe specific to RSPaV-1. As shown in FIG. 7, the Southern blot hybridization confirmed the results of the RT-PCR in each of the tested samples. Specifically, cDNA fragments amplified by RT-PCR from 16 selected RT-PCR positive samples hybridized with the probe.

Example 18 Constructing Expression Systems, Expression of a Fusion Protein Containing the RSPaV-1 Coat Protein, Production of Antibodies Against the Fusion Protein and Their Use in Detecting RSPaV-1 from Grapevines

The coat protein gene (SEQ. ID. No. 10) of RSPaV-1 was cloned into the EcoRI and HindIII sites of the polylinker region of a protein expression vector pMAL-c2 which, upon induction by inducer IPTG, produces a fusion protein containing maltose binding protein (MBP) and the coat protein of RSPaV-1. The fusion protein of expected size (ca. 71 KDa) was produced in E. coli bacteria after induction with IPTG. This fusion protein was purified through affinity chromatography using an amylose column. Purified fusion protein was used as an antigen to immunize a rabbit (by subcutaneous injection along the back) with the following scheme:

-   -   first injection, 400 μg fusion protein in 0.5 ml column buffer         with Freund's complete adjuvant;     -   second injection, 100 μg of protein in 0.5 ml column buffer with         Freund's incomplete adjuvant; and     -   third injection, 100 μg of protein in 0.5 ml buffer with         Freund's incomplete adjuvant.         Blood containing the antibodies was collected 70 days after the         first injection. The antibodies were recovered and successfully         used in an enzyme linked immunoabsorbent assay to detect the         presence of virus particles (i.e., coat protein) of RSPaV-1 from         a variety of tissue types of grapevines infected with RSP.

The antibodies produced against the expressed RSPaV-1 coat protein, therefore, are useful in the identification of the particles associated with RSP disease of grapevines, in the purification of the particles of RSPaV-1, and in the development of a serological diagnosis for RSP in grapevine. The use of the antibodies is suitable for detecting different strains of RSPaV-1. Because the coat proteins for strains RSP47-4 and RSP158 have high amino acid identities with the coat protein of RSPaV-1, it is very likely that the antibodies raised against RSPaV-1 coat protein will also detect other strains. Antibodies can be used in an ELISA to assay rapidly a large number of samples, thus making commercial development and utilization of diagnostic kits possible.

Example—19 Transformation of Grapevines with a Vector Containing RSPaV-1 Coat Protein Gene and Analysis of Transgenic Grapevines for Resistance to RSP

The DNA molecule coding for the RSPaV-1 coat protein (e.g., SEQ. ID. No. 10) was cloned into a pEPT8 plant expression vector that contains the double 35S enhancer at restriction sites SalI and BamHI. The resulting recombinant plasmid, designated pEPT8/RSPaV-1 coat protein, was then cloned into the plant transformation vector pGA482G, which has resistance genes to gentamycin and tetracycline as selection markers. The resultant pGA482G containing pEPT8/RSPaV-1 CP was used to transform grapevines using the Agrobacterium method.

The rootstock Vitis rupestris Scheele St. George was used in genetic transformation. Anthers were excised aseptically from flower buds. The pollen was crushed on a microscope slide with acetocarmine to observe the cytological stage (Bouquet et al., “Influence du Gentype sur la Production de cals: Dembryoides et Plantes Entieres par Culture Danthers in vitro dans le Genre Vitis,” C. R. Acad. Sci. Paris III 295:560-74 (1982), which is hereby incorporated by reference). This was done to determine which stage was most favorable for callus induction.

Anthers were plated under aseptic condition at a density of 40 to 50 per 9 cm diameter Petri dish containing MSE. Plates were cultured at 28° C. in the dark. After 60 days, embryos were induced and transferred to hormone-free medium (HMG) for differentiation. Torpedo stage embryos were transferred to MGC medium yo promote embryo germination. Cultures were maintained in the dark at 26-28° C. and transferred to fresh medium at 3-4 week intervals. Elongated embryos were transferred to rooting medium (5-8 embryos per jar). The embryos were grown in a tissue culture room at 25° C. with a daily 16 h photoperiod (76 μmol. s) to induce shoot and root formation. After plants developed roots, they were transplanted to soil in the greenhouse.

The protocols used for transformation were modified from those described by Scorza et al., “Transformation of Grape (Vitis vinifera L.) Zygotic-Derived Somatic Embryos and Regeneration of Transgenic Plants,” Plant Cell Rpt. 14:589-92 (1995), which is hereby incorporated by reference. Overnight cultures of Agrobacterium strain C58Z707 or LBA4404 were grown in LB medium at 28° C. in a shaking incubator. Bacteria were centrifuged for 5 minutes at 3000-5000 rpm and re-suspended in MS liquid medium (OD 1.0 at A600 nm). Calli with embryos were immersed in the bacterial suspension for 15-30 minutes, blotted dry, and transferred to HMG medium with or without acetosyringone (100 μM). Embryogenic calli were co-cultivated with the bacteria for 48 h in the dark at 28° C. The plant material was then washed in MS liquid plus cefotaxime (300 mg/ml) and carbenicillin (200 mg/ml) 2-3 times. To select transgenic embryos, the material was transferred to HMG medium containing either 20 or 40 mg/L kanamycin, 300 mg/L cefotaxime, and 200 mg/L carbenicillin. Alternatively, after co-cultivation, embryogenic calli were transferred to initiation MSE medium containing 25 mg/l kanamycin plus the same antibiotics listed above. All plant materials were incubated in continuous darkness at 28° C. After growth on selection medium for 3 months, embryos were transferred to HMG or MGC without kanamycin to promote elongation of embryos. They were then transferred to rooting medium without antibiotics. Non-transformed calli were grown on the same media with and without kanamycin to verify the efficiency of the kanamycin selection process.

The X-gluc (5-bromo-4-chloro-3-indoyl-β-glucuronidase) histochemical assay was used to detect GUS (β-glucuronidase) activity in embryos and plants that were transformed with constructs containing the GUS gene that survived kanamycin selection. All propagated plants were screened using an enzyme linked immunoabsorbent assay (ELISA) system (5 Prime-3 Prime, Boulder, Co.) to detect the NPTII (neomycin phosphotransferase II) protein in leaf extracts. ELISA tests with respective coat protein (CP)-specific antibodies were used to assay for CP. ELISA results were read on an SLT Spectra ELISA reader (Tecan U.S. Inc., Research Triangle Park, N.C.) 15-60 minutes after the substrate was added.

PCR analysis was carried out to detect the presence of transgene sequences in grape plants. Genomic DNA was isolated from transformed and non-transformed grape plants according to the method of Lodhi et al., “A Simple and Efficient Method for DNA Extraction from Grapevine Cultivars and Vitis Species,” Plant Mol. Biol. Rpt. 12:6-13 (1994), which is hereby incorporated by reference. Primer sets included those of specific primers to the transgene. DNA was initially denatured at 94° C. for 3 minutes, then amplified by 35 cycles of 1 minute at 94° C. (denaturing), 1 minute at 52° C. (annealing), and 2 minutes at 72° C. (polymerizing). Reaction samples were directly loaded and electrophoresed in 1.5% agarose gels.

Southern analysis of transformants was accomplished by extracting genomic DNA from young leaves of transformed and non-transformed plants (3309C) as described above. DNA (10 μg) was digested with the restriction enzyme Bgl II, electrophoresed on a 0.8% agarose gel in TAE buffer and transferred to a Genescreen Plus membrane by capillary in 10×SSC. A probe was prepared by random primer labeling of a PCR amplified gene coding sequence with radioisotope ³²P-dATP (Dupont, NEN). Pre-hybridization and hybridization steps were carried out at 65° C. following the manufacturer's instruction. The autoradiograph was developed after overnight exposure.

Although the invention has been described in detail for the purposes of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims. 

1. An isolated protein or polypeptide corresponding to a protein or polypeptide of a Rupestris stem pitting associated virus. 2-17. (cancelled)
 18. An isolated DNA molecule encoding a protein or polypeptide according to claim
 1. 19-34. (cancelled)
 35. A host cell transformed with a heterologous DNA molecule according to claim
 18. 36. The host cell according to claim 35, wherein the host cell is selected from a group consisting of Agrobacterium vitis and Agrobacterium tumefaciens.
 37. The host cell according to claim 35, wherein the host cell is a grape cell.
 38. The host cell according to claim 35, wherein the protein or polypeptide is selected from a group consisting of a replicase, a coat protein, and a protein of a triple gene block.
 39. A transgenic Vitis scion cultivar or rootstock cultivar comprising the DNA molecule according to claim
 18. 40. A transgenic Vitis scion cultivar or rootstock cultivar according to claim 39, wherein the protein or polypeptide is selected from a group consisting of a replicase, a coat protein, and a protein of a triple gene block. 41-63. (cancelled). 